CN103946371A - Method for secretory production of protein - Google Patents

Method for secretory production of protein Download PDF

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CN103946371A
CN103946371A CN201280054079.5A CN201280054079A CN103946371A CN 103946371 A CN103946371 A CN 103946371A CN 201280054079 A CN201280054079 A CN 201280054079A CN 103946371 A CN103946371 A CN 103946371A
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protein
rod
gene
type bacterium
bacterium
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CN103946371B (en
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松田吉彦
板屋宽
菊池庆实
别府春树
J.A.V.乔曼塔斯
E.A.库塔科瓦
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Ajinomoto Co Inc
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium

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Abstract

A method for secretory production of a heterologous protein is provided by developing a novel technique for improving ability of a coryneform bacterium to produce a heterologous protein by secretory production. By utilizing a coryneform bacterium having an ability to produce a heterologous protein by secretory production which has been modified so that activity of a penicillin-binding protein is reduced and in which activity of a cell surface layer protein has been reduced as an expression host, a heterologous protein is produced by secretory production.

Description

For method for secretory generation of protein
Technical field
The present invention relates to the rod-like stem bacterial type bacterium that heterologous protein is produced in efficient secretion, and for secreting the method that produces heterologous protein.
Background technology
About producing heterologous protein with microorganism secretion, report up to now and utilized the secretion such as filamentous fungus (non-patent document 3 and 4) of bacillus (Bacillus bacterium) (non-patent document 1), methyl alcohol assimilation yeast (methanol-assimilating yeast), pichia pastoris phaff (non-patent document 2), Eurotium to produce heterologous protein.
Also someone attempts producing heterologous protein with the secretion of rod-like stem bacterial type bacterium.About producing heterologous protein with the secretion of rod-like stem bacterial type bacterium, there is corynebacterium glutamicum (Corynebacterium glutamicum for report, below also be abbreviated as C.glutamicum) secretion nuclease and esterase (patent document 1, non-patent document 5), secretory protein enzyme is as bacillus subtilis protein matter enzyme (non-patent document 6), utilize the signal peptide of rod-like stem bacterial type bacterial cell surface layer protein PS1 and PS2 (also referred to as CspB) to carry out secretory protein (patent document 2), use the signal peptide of PS2 (CspB) to carry out Fibronectin Secretion conjugated protein (non-patent document 7), utilize the cell surface layer protein PS2 (CspB) of rod-like stem bacterial type bacterium and the signal peptide of SlpA (also referred to as CspA) to secrete protransglutaminase (protransglutaminase) (patent document 3), use variant excretory system secretory protein (patent document 4), with variant bacterial strain secretion protransglutaminase (patent document 5), utilize Tat dependent signals peptide to carry out secretory protein (patent document 6) etc.
There is multiple proteins to be considered as the protein producing for secretion.But, for example, about producing heterologous protein, the report that not yet has the secretion of any polymer protein being formed by multiple subunits (antibody associated molecule) to produce with the secretion of rod-like stem bacterial type bacterium.
Penicillin-binding protein (PBP) is the general name of the protein of being combined with beta-lactam type microbiotic, suppresses their enzyme function with the antibiotic combination of beta-lactam type.PBP is generally membrane bound protein, and they are considered to the synthetic of bacteria cell wall to be absolutely necessary.PBP is divided into high molecular PBP (HMW-PBP) and lower molecular weight PBP (LMW-PBP) according to its molecular weight.Wherein, HMW-PBP is further divided into category-A high molecular PBP (category-A HMW-PBP) and category-B high molecular PBP (category-B HMW-PBP).Category-A HMW-PBP possesses the active territory of the transpeptidase with transpeptidase activity for crosslinked peptidoglycan part, and for form the active territory of the transglycosylase with transglycosylase activity of polysaccharide chain from disaccharides from disaccharides; And category-B HMW-PBP only has the active territory of transpeptidase.
Discovery about the PBP of corynebacterium glutamicum has a detailed description in non-patent document 8,9 etc.In corynebacterium glutamicum, 9 kinds of PBP homologues are at least found up to now.Wherein 5 kinds is HMW-PBP, comprises two kinds of category-A HMW-PBP (PBP1a, PBP1b) and three kinds of category-B HMW-PBP (FstI, PBP2a, PBP2b).The category-A HMW-PBP of known corynebacterium glutamicum is responsible for the factor that cell stretches, and category-B HMW-PBP is the factor of being responsible for the peptidoglycan that forms septum wall in the time of cell fission.
Cell surface layer protein is the protein that forms bacterium and archeobacteria cell surface layer (S-layer).As the cell surface layer protein of rod-like stem bacterial type bacterium, known have the PS1 of corynebacterium glutamicum and a SlpA (CspA) (patent document 2) of PS2 (CspB) (non-patent document 10), C.stationis etc.For example, about PS2 (CspB), reported the aminoacid sequence (non-patent document 11) of 28 kinds of corynebacterium glutamicum bacterial strain CspB homologues.As mentioned above, the signal peptide of the cell surface layer protein of rod-like stem bacterial type bacterium is used to secretion and produces protein (patent document 2,3 etc.).
But, it be unclear that about reducing the active of penicillin-binding protein and/or reducing the activity of cell surface layer protein and secrete the relation producing between heterologous protein.
Prior art reference
Patent document
Patent document 1: U.S. Patent No. 4,965,197
Patent document 2: Japanese Patent JP-A 6-502548
Patent document 3: Japanese Patent 4320769
Patent document 4: Japanese patent laid-open 11-169182
Patent document 5: Japanese Patent 4362651
Patent document 6: Japanese Patent 4730302
Non-patent document
Non-patent document 1:Microbiol.rev., 57,109-137 (1993)
Non-patent document 2:Biotechnol., 11,905-910 (1993)
Non-patent document 3:Biotechnol., 6,1419-1422 (1988)
Non-patent document 4:Biotechnol., 9,976-981 (1991)
Non-patent document 5:J.Bacteriol., 174,1854-1861 (1992)
Non-patent document 6:Appl.Environ.Microbiol., 61,1610-1613 (1995)
Non-patent document 7:Appl.Environ.Microbiol., 63,4392-4400 (1997)
Non-patent document 8:Mol.Microbiol., 66,643-57 (2007)
Non-patent document 9:Antonie Van Leeuwenhoek, 94,99-109 (2008)
Non-patent document 10:Mol.Microbiol., 9,97-109 (1993)
Non-patent document 11:J Biotechnol., 112,177-193 (2004)
Summary of the invention
The object that the present invention will realize
A target of the present invention is a kind of new technology that produces the ability of heterologous protein for improving the secretion of rod-like stem bacterial type bacterium of exploitation, the rod-like stem bacterial type bacterium that provides thus secretion to produce heterologous protein, and use this bacterium secretion to produce the method for heterologous protein.
The means of realize target
The inventor has carried out research in many ways for realizing aforementioned target, they find result, in the method for utilizing rod-like stem bacterial type bacterium as expressive host generation heterologous protein, by the gene of disappearance rod-like stem bacterial type bacterial identification penicillin-binding protein PBP1a and the gene of Codocyte surface layer protein CspB, can improve the secretion of rod-like stem bacterial type bacterium and produce the ability of heterologous protein, and realize the present invention.
Therefore, the invention provides as follows:
[1] there is a rod-like stem bacterial type bacterium that secretes the ability that produces heterologous protein,
This bacterium has reduced the activity of penicillin-binding protein through modifying, and in this bacterium, has reduced the activity of cell surface layer protein, and
Wherein said penicillin-binding protein is the protein with following character: in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of described heterologous protein increases than the secretion generation of observing in the bacterial strain of unmodified.
[2] rod-like stem bacterial type bacterium as above, thus its through modification by reduction encode described penicillin-binding protein gene expression or destroy this gene and reduced the activity of described penicillin-binding protein.
[3] rod-like stem bacterial type bacterium as above, wherein said penicillin-binding protein is PBP1a.
[4] rod-like stem bacterial type bacterium as above, wherein said penicillin-binding protein is (A) or (B) protein of middle definition below:
(A) there is the protein of the aminoacid sequence of SEQ ID NO:82,
(B) there is the aminoacid sequence of the SEQ ID NO:82 of the replacement, disappearance, insertion or the interpolation that comprise 1-10 amino-acid residue, and there is the protein of following character: in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of described heterologous protein increases than the secretion generation of observing in the bacterial strain of unmodified.
[5] rod-like stem bacterial type bacterium as above, thus its through modification by reduction encode described cell surface layer protein gene expression or destroy this gene and reduced the activity of described cell surface layer protein.
[6] rod-like stem bacterial type bacterium as above, wherein said cell surface layer protein is CspB.
[7] rod-like stem bacterial type bacterium as above, wherein said cell surface layer protein is (A) or (B) protein of middle definition below:
(A) there is the protein of SEQ ID NO:98 aminoacid sequence,
(B) there is the aminoacid sequence of the SEQ ID NO:98 of the replacement, disappearance, insertion or the interpolation that comprise 1-10 amino-acid residue, and there is the protein of following character: in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of described heterologous protein increases than the secretion generation of observing in the bacterial strain of unmodified.
[8] rod-like stem bacterial type bacterium as above, it is the bacterium that belongs to corynebacterium (Corynebacterium) or brevibacterium sp (Brevebacterium).
[9] rod-like stem bacterial type bacterium as above, it is corynebacterium glutamicum (Corynebacterium glutamicum).
[10] rod-like stem bacterial type bacterium as above,
Wherein said rod-like stem bacterial type bacterium has the gene construct for heterologous protein described in secreting, expressing, and
Wherein said gene construct comprises: the promoter sequence of bringing into play function in described rod-like stem bacterial type bacterium, be connected to this promoter sequence downstream, be coded in described rod-like stem bacterial type bacterium and bring into play the nucleotide sequence of the signal peptide of function, and be connected to nucleotide sequence nucleotide sequence downstream, that encode described heterologous protein of this coded signal peptide.
[11] rod-like stem bacterial type bacterium as above, wherein said heterologous protein is antibody associated molecule.
[12] rod-like stem bacterial type bacterium as above, wherein this antibody associated molecule is by being selected from Fab, F (ab') 2, one or more protein in Fc-fused protein and scFv form.
[13], for generation of a method for heterologous protein, it comprises cultivates rod-like stem bacterial type bacterium as above, and collects the heterologous protein that secretion produces.
Accompanying drawing summary
[Fig. 1] Fig. 1 is presented at the photo of expressing the result of the reduced form SDS-PAGE carrying out after Herceptin Fab fragment H sequence in YDK010 bacterial strain (parent strain) and YDK010 Δ PBP1a bacterial strain.
[Fig. 2] Fig. 2 is presented at the photo of expressing the result of the reduced form SDS-PAGE carrying out after the Fab fragment H sequence of Herceptin in YDK010 bacterial strain (parent strain), YDK010 Δ PBP1a bacterial strain and YDK010 Δ PBP1b bacterial strain.
[Fig. 3] Fig. 3 is presented at the photo of expressing the result of the reduced form SDS-PAGE carrying out after the Fab fragment H sequence of Herceptin in YDK010 bacterial strain (parent strain) and YDK010 Δ PBP1a bacterial strain.
[Fig. 4] Fig. 4 is presented at the photo of expressing the result of the reduced form SDS-PAGE carrying out after Herceptin Fab fragment L sequence in YDK010 bacterial strain (parent strain) and YDK010 Δ PBP1a bacterial strain.
[Fig. 5] Fig. 5 is the photo that is presented at the result of the non-reduced type SDS-PAGE carrying out after coexpression Herceptin Fab fragment H sequence and L sequence in YDK010 bacterial strain (parent strain) and YDK010 Δ PBP1a bacterial strain.
[Fig. 6] Fig. 6 is presented at the photo of expressing the result of the Western trace carrying out after Herceptin F (ab') 2 fragments in YDK010 bacterial strain (parent strain) and YDK010 Δ PBP1a bacterial strain.
[Fig. 7] Fig. 7 is presented at the photo of expressing the result of the Western trace carrying out after Herceptin Fc fragment in YDK010 bacterial strain (parent strain) and YDK010 Δ PBP1a bacterial strain.
[Fig. 8] Fig. 8 is the figure that is presented at the expression amount of the protransglutaminase of expressing in YDK010 bacterial strain (parent strain) and YDK010 Δ PBP1a bacterial strain.
[Fig. 9] Fig. 9 is presented at the result of expressing the reduced form SDS-PAGE carrying out after anti-digoxin single-chain antibody in YDK010 bacterial strain (parent strain) and YDK010 Δ PBP1a bacterial strain.
[Figure 10] Figure 10 is presented at the photo of expressing the result of the non-reduced type SDS-PAGE carrying out after adalimumab (adalimumab) Fab (H & L) fragment in YDK010 bacterial strain (parent strain) and YDK010 Δ PBP1a bacterial strain.
[Figure 11] Figure 11 is presented at the photo of expressing the result of the non-reduced type SDS-PAGE carrying out after Herceptin Fab (H & L) fragment in ATCC13869 bacterial strain (parent strain), ATCC13869 Δ CspB bacterial strain, ATCC13869 Δ PBP1a bacterial strain and ATCC13869 Δ CspB Δ PBP1a bacterial strain.
Implement pattern of the present invention
<1> rod-like stem bacterial type of the present invention bacterium
The invention provides a kind of rod-like stem bacterial type bacterium of the ability with secretion generation heterologous protein, this bacterium has reduced the activity of penicillin-binding protein through modifying, and has wherein reduced the activity (hereinafter also referred to as " bacterium of the present invention " or " rod-like stem bacterial type bacterium of the present invention ") of cell surface layer protein.
In the present invention, protein is that protein is transported out bacterial cell (extracellular transhipment) by the meaning of " secretion ".Protein is comprised that by " secretion " all molecules of protein are finally present in the situation in substratum with completely free form natch, also all molecules that comprise protein are present in the situation in cell surface layer, and the part molecule of protein is present in substratum, and rest part molecule is present in the situation in cell surface layer.
That is to say, in the present invention, " secretion produces the ability of heterologous protein " refers to the following ability of bacterium of the present invention, when cultivate this bacterium in substratum time, it can be secreted into heterologous protein in substratum or cell surface layer, and make it in the accumulation of substratum or cell surface layer, be able to the degree of collecting this heterologous protein from substratum or cell surface layer.About accumulation volume, for example, the accumulation volume in substratum can be preferably 10 μ g/L or more, more preferably 1mg/L or more, particularly preferably 100mg/L or more, still more preferably 1g/L or more.In addition, about accumulation volume, for example, accumulation volume in cell surface layer can be following degree, if when cell surface layer is collected heterologous protein and be suspended in the medium liquid of same volume, in suspension, the concentration of heterologous protein is preferably 10 μ g/L or more, more preferably 1mg/L or more, particularly preferably 100mg/L or more.In addition, in the present invention, " protein " that term secretion produces refers to and comprises the protein concept that is known as peptide or polypeptide.
In the present invention, " heterologous protein " refers to that be the protein of external source for the rod-like stem bacterial type bacterium of this protein of expression and secretion.Heterologous protein can be that for example, from the protein of microorganism, from the protein of plant, from the protein of animal, from viral protein, or even aminoacid sequence is the protein of artificial design.Heterologous protein can be monomeric protein or polymer protein.Polymer protein refers to the protein that can be used as the polymer existence being made up of two or more subunits.In polymer, subunit can connect by for example disulfide linkage of covalent linkage, be connected with hydrophobic interaction by the non-covalent for example hydrogen bond of building, or being connected by these.Polymer preferably comprises one or more intermolecular disulfide bonds.Polymer can be the same polymer being made up of the subunit of single kind, or can be the heteromultimeric being made up of two or more subunits.In the situation that polymer is heteromultimeric, be heterologous protein as long as have at least one subunit in the subunit of formation heteromultimeric.That is to say, all subunits can be allos, or only some subunit is allos.Although heterologous protein may be natural be secreted protein, or natural be non-secreted protein, preferred natural is secreted protein.The specific examples of " heterologous protein " will be mentioned below.
The heterologous protein producing can be made up of the protein of single type, or is made up of the protein of two or more types.And, in the time that heterologous protein is heteromultimers, the wherein subunit of a type can be only produced, or the subunit of two or more types can be produced.That is to say, " secretion of heterologous protein produces " comprises that secretion produces all subunits that form target heterologous protein, also comprises that only secretion produces a part of subunit that forms target heterologous protein.
In the present invention, rod-like stem bacterial type bacterium is aerobic Gram-positive bacillus (bacilli), comprises corynebacterium bacterium, brevibacterium sp bacterium, Microbacterium bacterium etc.Rod-like stem bacterial type bacterium comprises the bacterium (Int.J.Syst.Bacteriol., 41,255 (1991)) that is classified as brevibacterium sp in the past but be integrated into now corynebacterium.Rod-like stem bacterial type bacterium also comprises and had previously been classified into corynebacterium ammoniagenes but had now been reclassified the bacterium (Int.J.Syst.Evol.Microbiol., 60,874-879 (2010)) in Corynebacterium stationis by 16S rRNA nucleotide sequence analysis etc.Use the advantage of rod-like stem bacterial type bacterium to comprise: they with conventionally produce protedogenous fungi for secreting, yeast is compared with genus bacillus, the protein secreting amount of itself is few, and the purge process of the heterologous protein therefore secretion being produced can be simplified or omit; They can well grow in the simple culture media that contains polysaccharide, ammonia, mineral salt etc., are therefore excellent at aspects such as culture medium cost, cultural method, cultivation productivity; Etc..
The specific examples of these rod-like stem bacterial type bacteriums comprises following species:
Have a liking for etheric acid coryneform bacteria (Corynebacterium acetoacidophilum)
Acetylglutamate coryneform bacteria (Corynebacterium acetoglutamicum)
Corynebacterium alkanolyticum (Corynebacterium alkanolyticum)
Corynebacterium callunae (Corynebacterium callunae)
Corynebacterium glutamicum (Corynebacterium glutamicum)
Corynebacterium lilium (Corynebacterium lilium)
The molasses coryneform bacteria of dwelling (Corynebacterium melassecola)
Thermophilic corynebacterium ammoniagenes (Corynebacterium thermoaminogenes) (Corynebacterium efficiens)
Man of great strength's coryneform bacteria (Corynebacterium herculis)
Bifid tyrothricin (Brevibacterium divaricatum)
Brevibacterium flavum (Brevibacterium flavum)
Brevibacterium?immariophilum
Brevibacterium (Brevibacterium lactofermentum) (corynebacterium glutamicum)
Pink tyrothricin (Brevibacterium roseum)
Brevibacterium saccharolyticum (Brevibacterium saccharolyticum)
Sulphur is grown tyrothricin (Brevibacterium thiogenitalis)
Corynebacterium ammoniagenes (Corynebacterium ammoniagenes) (Corynebacterium stationis)
Brevibacterium albus (Brevibacterium album)
Polygonal tyrothricin (Brevibacterium cerinum)
Have a liking for ammonia microbacterium (Microbacterium ammoniaphilum)
The specific examples of these rod-like stem bacterial type bacteriums comprises following bacterial strain:
Have a liking for acetic acid corynebacteria A TCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium alkanolyticum ATCC21511
Corynebacterium callunae ATCC15991
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060, ATCC13869, FERM BP-734
Corynebacterium lilium ATCC15990
The molasses corynebacteria A of dwelling TCC17965
Thermophilic corynebacterium ammoniagenes AJ12340 (FERM BP-1539)
Man of great strength's corynebacteria A TCC13868
Bifid tyrothricin ATCC14020
Brevibacterium flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205)
Brevibacterium?immariophilum?ATCC14068
Brevibacterium lactofermentus ATCC13869
Pink tyrothricin ATCC13825
Brevibacterium saccharolyticum ATCC14066
Sulphur is grown tyrothricin ATCC19240
Brevibacterium ammoniagenes (Corynebacterium stationis) ATCC6871, ATCC6872
Brevibacterium albus ATCC15111
Polygonal tyrothricin ATCC15112
Have a liking for ammonia microbacterium ATCC15354
These bacterial strains can obtain from for example American type culture collection (ATCC) (address: 12301Parklawn Drive, Rockville, Maryland20852, P.O.Box1549, Manassas, VA20108, United States of America).That is to say, each bacterial strain all has a unique accession number (http://www.atcc.org/), and can be by using this accession number to order.The accession number of each bacterial strain is listed in the catalogue of ATCC.
Especially, corynebacterium glutamicum AJ12036 bacterial strain (FERM BP-734), it separates and obtains from wild strain corynebacterium glutamicum ATCC13869 as anti-streptomycin (Sm) mutant strain, this bacterial strain prediction has sudden change in the functional gene of being responsible for protein secreting, and show that high protein secreting produces ability: in the protein accumulation amount under optimal culture condition, up to 2-3 doubly, therefore it is preferably as host bacteria compared with parent strain (wild type strain).It (is now the biological preservation of independent administrative legal person's products assessment technique basal disc organization's patent center that AJ12036 bacterial strain (FERM BP-734) is deposited in life engineering technical institute of Ministry of International Trade and Industry science and technology institute on March 26th, 1984 as international preservation at first, AIST Tsukuba Central6,1-1, Higashi1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566,, and be given accession number FERM BP-734 Japan).
And, the bacterial strain that can select protein secreting generation ability to strengthen from the rod-like stem bacterial type bacterium obtaining by use mutafacient system or genetic recombination method using rod-like stem bacterial type bacterium as above as parent strain, and as host.For example, utilizing uv-radiation or chemical mutagen to process after parent strain as N-methyl-N'-nitrosoguanidine, the bacterial strain that can select protein secreting generation ability to strengthen.
Further, if will make it not produce bacterial strain that cell surface layer protein obtains as host by modifying above-mentioned bacterial strains, the purifying of secreting the heterologous protein in substratum will become easily, be therefore particularly preferred.These modifications can realize by utilizing mutagenesis or genetic recombination to introduce sudden change to the expression regulation district on the coding region of cell surface layer protein or its karyomit(e).Thereby the example of being modified the rod-like stem bacterial type bacterium that does not produce cell surface layer protein comprises corynebacterium glutamicum YDK010 bacterial strain (WO2004/029254), it is the cell surface layer protein PS2 deficient strain of corynebacterium glutamicum AJ12036 bacterial strain (FERM BP-734).
The rod-like stem bacterial type bacterium with the ability of secretion generation heterologous protein can, by import the gene construct for secreting, expressing heterologous protein in rod-like stem bacterial type bacterium as above, obtain thereby make bacterium carry this construct.That is to say, bacterium of the present invention has the gene construct for secreting, expressing heterologous protein.This " for gene construct of secreting, expressing heterologous protein " and its method of importing will below make an explanation.
Bacterium of the present invention can be modified by the rod-like stem bacterial type bacterium that produces heterologous protein ability to having secretion, makes the activity of penicillin-binding protein and the activity decreased of cell surface layer protein and obtains.Bacterium of the present invention can also, by rod-like stem bacterial type bacterium is modified, make the activity of penicillin-binding protein and the activity decreased of cell surface layer protein, and then gives the ability of its secretion generation heterologous protein and obtain.And, bacterium of the present invention can also be modified by the rod-like stem bacterial type bacterium that the activity of cell surface layer protein own has been lowered, thereby make this bacterium there is the ability of heterologous protein of generation, and make the activity decreased of its penicillin-binding protein and obtain.In the present invention, for the modification and the giving of ability that build bacterium of the present invention can be carried out according to any order.Bacterium of the present invention can be to produce the bacterium of heterologous protein from secreting, and reduces by modifying the bacterium that the activity of its penicillin-binding protein and/or the activity of cell surface layer protein obtain.In addition, bacterium of the present invention can also be the bacterium that produces heterologous protein (even when its contain while being useful on the gene construct of secreting, expressing heterologous protein) from secreting, reduce the activity of its penicillin-binding protein and/or the activity of cell surface layer protein by modifying, and the active modification of the activity of this reduction penicillin-binding protein and/or cell surface layer protein causes this bacterium to become can secreting generation heterologous protein, thus the bacterium obtaining.In addition, bacterium of the present invention can further be modified, and has and the expression of the gene of the protein in the region of the motif homology of metal titanium enzyme thereby increased the gene of coding metallopeptidase or coding.
Hereinafter, will make an explanation to penicillin-binding protein and its encoding gene.
Usually, penicillin-binding protein (PBP) refers to such protein, and it is combined with beta-lactam type microbiotic, and its enzyme function is suppressed by being combined with beta-lactam type microbiotic.Penicillin-binding protein comprises high molecular PBP (HMW-PBP) and lower molecular weight PBP (LMW-PBP).High molecular PBP comprises category-A high molecular PBP (category-A HMW-PBP) and category-B high molecular PBP (category-B HMW-PBP).Category-A HMW-PBP has the active territory of a transpeptidase, and it has transpeptidase activity, for crosslinked peptidoglycan part; With the active territory of a transglycosylase, it has transglycosylase activity, for forming polysaccharide chain from disaccharides.Category-B HMW-PBP has the active territory of a transpeptidase.For example, for corynebacterium glutamicum, PBP1a and PBP1b can think category-A HMW-PBP.For corynebacterium glutamicum, FtsI, PBP2a and PBP2b can think category-B HMW-PBP.
In the present invention, the activity of protein as described below is lowered: it is penicillin-binding protein, and there is in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered the character of the secretion generation of heterologous protein increase compared with secretion generation seen in not modified bacterial strain.About such penicillin-binding protein, for example, be selected from PBP1a, category-B HMW-PBP and LMW-PBP person and be preferred, be selected from PBP1a and category-B HMW-PBP person is preferred, PBP1a is particularly preferred.
" in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered; the character of the secretion generation of heterologous protein increase compared with secretion generation seen in not modified bacterial strain " refers to following character, if the activity of this protein is lowered in rod-like stem bacterial type bacterium, this rod-like stem bacterial type bacterium is endowed following ability: compared with observing in not modified for example wild type strain of bacterial strain or parent strain, the amount that secretion produces heterologous protein is larger.Do not have special restriction although secrete the increase degree of the amount of the heterologous protein of generation, as long as secretion produce heterologous protein amount than in not modified bacterial strain, observe more greatly, but the amount of the heterologous protein that secretion produces is than can meaning more greatly of observing in not modified bacterial strain, for example, in the amount accumulating in substratum and/or cell surface layer, the amount of the heterologous protein that secretion produces is than the amount of observing in not modified bacterial strain greatly preferably 10% or more, more preferably 20% or more, particularly preferably 30% or more, further preferably 100% or more.In addition, " amount of the heterologous protein that secretion produces is larger than the amount of observing in not modified bacterial strain " can represent: in the time that the not concentrated culture supernatant of the bacterial strain to not modified is carried out SDS-PAGE and dyes with CBB, heterologous protein can not be detected, and in the time that the not concentrated nutrient solution supernatant of the bacterial strain to through modifying carries out SDS-PAGE and dyes with CBB, heterologous protein can be detected.
In addition, about penicillin-binding protein, " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered; the character of the secretion generation of heterologous protein increase compared with secretion generation seen in not modified bacterial strain " comprises following character: if reduce the activity of this protein in the bacterial strain not reducing at cell surface layer protein active, the ability that this bacterial strain secretion produces heterologous protein does not increase; If but in the lowered bacterial strain of cell surface layer protein active, reduce the activity of this protein, the ability that this bacterial strain secretion produces heterologous protein increases.
Whether protein has " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of heterologous protein increases compared with secretion generation seen in not modified bacterial strain " character, can confirm by the following method: the bacterial strain that is subordinated to rod-like stem bacterial type bacterium makes protein active reduce to prepare bacterial strain by modification, measure the secretion generation of the heterologous protein of observing when cultivate the bacterial strain of this process modification in substratum time, and this amount and the secretion generation of heterologous protein when cultivate not modified bacterial strain (unmodified bacterial strain) in substratum time are compared.
The Cgl0278 gene of coding corynebacterium glutamicum ATCC13032PBP1a protein is corresponding to the complementary sequence of the genome sequence 294001-296388 bit sequence of logining with GenBank accession number BA000036 (VERSION BA000036.3GI:42602314) in ncbi database.In addition, the GenBank accession number of corynebacterium glutamicum ATCC13032PBP1a protein is NP_599531 (version NP_599531.1GI:19551529, locus_tag=" NCgl0274 ").The nucleotide sequence of corynebacterium glutamicum ATCC13032Cgl0278 gene and by the aminoacid sequence of the PBP1a protein of this genes encoding respectively as shown in SEQ ID NO:81 and 82.
Because the nucleotide sequence of the gene of Renicillin binding protein matter can change along with the difference of rod-like stem bacterial type bacterium institute's species and bacterial strain, so the gene of Renicillin binding protein matter can be the variant of foregoing nucleotide sequence, as long as this genes encoding has the protein of the character of " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of heterologous protein increases compared with secretion generation seen in not modified bacterial strain ".In addition, the variant of Cgl0278 gene comprises the homologue of this gene.The homologue of Cgl0278 gene can be retrieved or FASTA retrieval by BLAST, use the wild-type Cgl0278 gene of aforementioned corynebacterium glutamicum easily to obtain from public database as search sequence, the oligonucleotide that karyomit(e) that can also be by using rod-like stem bacterial type bacterium is prepared as template with according to known sequence (example described above those) carries out PCR as primer and obtains.
The gene of Renicillin binding protein matter can be the gene that coding has the protein of the aforementioned aminoacid sequence of the replacement, disappearance, insertion or the interpolation that comprise or several amino-acid residues on one or more positions, as long as this genes encoding has the protein of the character of " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of heterologous protein increases compared with secretion generation seen in not modified bacterial strain ".In this case, with do not import that one or several amino-acid residue replace, disappearance, insert or the protein that adds compared with, the character of " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered; the secretion generation of heterologous protein increases compared with secretion generation seen in not modified bacterial strain " keeps 70% or more conventionally, preferably 80% or more, more preferably 90% or more.Although the number of " one or several " amino-acid residue can change according to the type of the position in protein three-dimensional structure or amino-acid residue, particularly, its number is preferably 1-20, more preferably 1-10, more more preferably 1-5.
Replacement, disappearance, insertion or the interpolation of aforementioned one or several amino-acid residues is the conservative sudden changes that keep the normal function of protein.The representative instance of conservative sudden change is conservative replacement.Conservative replacement refers to following sudden change, is die aromatischen Aminosaeuren if wherein replace site, replaces and betides between Phe, Trp and Tyr; If it is hydrophobic amino acid, betide between Leu, Ile and Val; If it is polare Aminosaeren, betide between Gln and Asn; If it is basic aminoacids, betide between Lys, Arg and His; If it is acidic amino acid, betide between Asp and Glu; If it is the amino acid with hydroxyl, betide between Ser and Thr.Particularly, being considered as the conservative replacement example replacing comprises, Ser or Thr replace Ala, Gln, His or Lys replace Arg, Glu, Gln, Lys, His or Asp replace Asn, Asn, Glu or Gln replace Asp, Ser or Ala replace Cys, Asn, Glu, Lys, His, Asp or Arg replace Gln, Gly, Asn, Gln, Lys or Asp replace Glu, Pro replaces Gly, Asn, Lys, Gln, Arg or Tyr replace His, Leu, Met, Val or Phe replace Ile, Ile, Met, Val, or Phe replaces Leu, Asn, Glu, Gln, His or Arg replace Lys, Ile, Leu, Val or Phe replace Met, Trp, Tyr, Met, Ile or Leu replace Phe, Thr or Ala replace Ser, Ser or Ala replace Thr, Phe or Tyr replace Trp, His, Phe or Trp replace Tyr, and Met, Ile or Leu replace Val.Further, the replacement of above-mentioned these amino-acid residues, disappearance, insertion, interpolation, inversion etc. comprise the abiogenous sudden change (mutant or variant) causing due to the individual diversity XOR species difference of the bacterium of this gene institute origin.
Further, the gene with above-mentioned these conservative sudden changes can be the gene of the following protein of coding, this protein and total coding aminoacid sequence show 80% or higher, preferably 90% or higher, more preferably 95% or higher, again more preferably 97% or higher, particularly preferably 99% or higher homology, and there is the character of " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of heterologous protein increases compared with secretion generation seen in not modified bacterial strain ".In addition,, in patent specification, " homology " can mean " identity ".
And, the gene of Renicillin binding protein matter can be such DNA, it can be under stringent condition be hybridized mutually with the probe of for example, preparing from known sequence (with part or all complementary sequence of the above-mentioned nucleotide sequence), and coding has the protein of the character of " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of heterologous protein increases compared with secretion generation seen in not modified bacterial strain "." stringent condition " refers to such condition, forms so-called specific hybrid body, and do not form nonspecific crossbred under this condition.The example of stringent condition comprises following condition, under this condition, the DNA of height homology is hybridized each other, for example be not less than 80% homology, preferably be not less than 90% homology, more preferably be not less than 95% homology, more preferably be not less than 97% homology, the DNA that is particularly preferably not less than 99% homology is hybridized each other, and homology can not hybridized each other lower than above-mentioned DNA, or typical Southern hybridization wash conditions, corresponding to 1xSSC, 0.1%SDS60 DEG C, preferably 0.1x SSC, 0.1%SDS60 DEG C, more preferably 0.1x SSC, under the salt concn and temperature of 0.1%SDS68 DEG C, clean 1 time, preferably 2 or 3 times.
For the probe of aforementioned hybridization can be and the part of the sequence of said gene complementation.Such probe can use the oligonucleotide prepared according to known sequence as primer, using the DNA fragmentation that contains this nucleotide sequence as template, is prepared by PCR.For example, when to use length be about 300bp DNA fragmentation is during as probe, the cleaning condition of hybridization can be, for example 50 DEG C, 2x SSC and 0.1%SDS.
In addition, the aforementioned explanation about gene and protein variant also can be applied to arbitrary protein matter after suitable variation, for example cell surface layer protein of the present invention and will secrete the heterologous protein of generation, and their gene of encoding.
Hereinafter, will make an explanation to cell surface layer protein and encoding gene thereof.
Cell surface layer protein is the protein that forms the cell surface layer (S-layer) of bacterium and archeobacteria.The example of the cell surface layer protein of rod-like stem bacterial type bacterium comprises the PS1 of corynebacterium glutamicum and the SlpA (also referred to as CspA) of PS2 (also referred to as CspB) and C.stationis.Among them, the preferably activity decreased of PS2.
The nucleotide sequence of corynebacterium glutamicum ATCC13869cspB gene and by the aminoacid sequence of the PS2 protein of this genes encoding respectively as shown in SEQ ID NO:97 and 98.
In addition, for example, have been reported (J Biotechnol., 112,177-193 (2004)) about the aminoacid sequence of the CspB homologue of 28 kinds of corynebacterium glutamicum bacterial strains.List these 28 kinds of corynebacterium glutamicum bacterial strains and the GenBank accession number (GenBank accession number in bracket show) of these cspB gene homologs in ncbi database below.
Corynebacterium glutamicum ATCC13058 (AY524990)
Corynebacterium glutamicum ATCC13744 (AY524991)
Corynebacterium glutamicum ATCC13745 (AY524992)
Corynebacterium glutamicum ATCC14017 (AY524993)
Corynebacterium glutamicum ATCC14020 (AY525009)
Corynebacterium glutamicum ATCC14067 (AY524994)
Corynebacterium glutamicum ATCC14068 (AY525010)
Corynebacterium glutamicum ATCC14747 (AY525011)
Corynebacterium glutamicum ATCC14751 (AY524995)
Corynebacterium glutamicum ATCC14752 (AY524996)
Corynebacterium glutamicum ATCC14915 (AY524997)
Corynebacterium glutamicum ATCC15243 (AY524998)
Corynebacterium glutamicum ATCC15354 (AY524999)
Corynebacterium glutamicum ATCC17965 (AY525000)
Corynebacterium glutamicum ATCC17966 (AY525001)
Corynebacterium glutamicum ATCC19223 (AY525002)
Corynebacterium glutamicum ATCC19240 (AY525012)
Corynebacterium glutamicum ATCC21341 (AY525003)
Corynebacterium glutamicum ATCC21645 (AY525004)
Corynebacterium glutamicum ATCC31808 (AY525013)
Corynebacterium glutamicum ATCC31830 (AY525007)
Corynebacterium glutamicum ATCC31832 (AY525008)
Corynebacterium glutamicum LP-6 (AY525014)
Corynebacterium glutamicum DSM20137 (AY525015)
Corynebacterium glutamicum DSM20598 (AY525016)
Corynebacterium glutamicum DSM46307 (AY525017)
Corynebacterium glutamicum 22220 (AY525005)
Corynebacterium glutamicum 22243 (AY525006)
Because the nucleotide sequence of the gene of Codocyte surface layer protein may change along with the difference of the kind under rod-like stem bacterial type bacterium or bacterial strain, so the gene of Codocyte surface layer protein can be the variant of foregoing nucleotide sequence, as long as genes encoding has the protein of the character of " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of heterologous protein increases compared with secretion generation seen in not modified bacterial strain ".For example, the gene of Codocyte surface layer protein can be the gene that coding has the protein of the aminoacid sequence of the replacement, disappearance, insertion or the interpolation that comprise or several amino-acid residues on or several position, as long as genes encoding has the protein of the character of " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of heterologous protein increases compared with secretion generation seen in not modified bacterial strain ".The aforementioned explanation about penicillin-binding protein and its gene of coding also can be applied to cell surface layer protein and encoding gene thereof after suitable variation.
In addition, about cell surface layer protein, " in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of heterologous protein increases compared with secretion generation seen in not modified bacterial strain " character comprise following character: if reduce the activity of protein in the bacterial strain not reducing in penicillin-binding protein activity, the ability of bacterial strain secretion generation heterologous protein can not increase, if but reduce the activity of protein in the active lowered bacterial strain of penicillin-binding protein, the ability of bacterial strain secretion generation heterologous protein can increase.
In the present invention, statement " activity of cell surface layer protein is lowered " thus comprise that rod-like stem bacterial type bacterium is through modifying the active situation of cell surface layer protein of having reduced, and the situation that the activity of cell surface layer protein is lowered inherently in rod-like stem bacterial type bacterium." situation that the activity of cell surface layer protein is lowered inherently in rod-like stem bacterial type bacterium " comprises that rod-like stem bacterial type bacterium lacks the situation of cell surface layer protein inherently.That is to say, the example of the rod-like stem bacterial type bacterium that cell surface layer protein active is lowered comprises the rod-like stem bacterial type bacterium that lacks inherently cell surface layer protein.The example of " rod-like stem bacterial type bacterium lacks the situation of cell surface layer protein inherently " comprises that rod-like stem bacterial type bacterium lacks the situation of the gene of Codocyte surface layer protein inherently.Statement " rod-like stem bacterial type bacterium lacks cell surface layer protein inherently ", can represent that this rod-like stem bacterial type bacterium lacks one or more and be selected from the protein of the cell surface layer protein of finding in other bacterial strain of this rod-like stem bacterial type bacterium institute species inherently.For example, " corynebacterium glutamicum lacks cell surface layer protein inherently " can represent that this corynebacterium glutamicum bacterial strain lacks one or more and be selected from the protein of the cell surface layer protein of finding in other corynebacterium glutamicum bacterial strain inherently,, for example, lack PS1 and/or PS2 (CspB).The rod-like stem bacterial type bacterium that lacks inherently cell surface layer protein comprises corynebacterium glutamicum ATCC13032, itself is cspB gene defection type.
Hereinafter, will the method that reduce protein active be made an explanation.
Statement " protein active is lowered " means, compares the activity decreased of target protein with not modified for example wild type strain of bacterial strain with parent strain, comprises the situation of active completely dissolve.Particularly, statement " protein active is lowered " means, compared with not modified bacterial strain, the molecule number of the protein of each cell reduces and/or the function of each protein molecule reduces.That is to say, about statement language " activity of protein is lowered ", the amount of transcribing (mRNA amount) of the gene that term " activity " can this protein of presentation code or the amount of this protein, and the catalytic activity of protein.In addition, the situation of " molecule number of the protein of each cell reduces " comprises protein non-existent situation at all.Further, the situation of " function of each protein molecule reduces " comprises the situation of the function completely dissolve of each protein molecule.
For the modification that reduces protein active can be passed through, for example, the expression that reduces protein coding gene realizes." minimizing genetic expression " is also referred to as " reduction genetic expression ".Reducing genetic expression can pass through, and for example, reduces and transcribes efficiency, reduces translation efficiency or its and combine to realize.The minimizing of genetic expression can be by the expression regulation sequence of modifying factor, and for example promotor and Shine-Dalgarno (SD) sequence, realizes.In the time modifying expression regulation sequence, preferably modify one or more Nucleotide of expression regulation sequence, more preferably two or more Nucleotide, particularly preferably three or more Nucleotide.And, part or all that can loss of expression regulating and controlling sequence.Reducing genetic expression can also realize by the factor of for example handling responsible expression regulation.The example of the factor of being responsible for expression regulation comprises, be responsible for transcribing or translating control small molecules (inductor, inhibitor etc.), be responsible for transcribing or translating control protein (transcription factor etc.), be responsible for transcribing or translating the nucleic acid (siRNA etc.) etc. of control.
For the modification that reduces protein active can also be realized by for example destroying protein coding gene.The destruction of gene can be realized by part or all of the coding region of this gene on deletion for example.And, can lack the whole gene the upstream and downstream sequence of gene on karyomit(e).The region lacking can be any region, and for example N-end regions, interior region or C end regions, as long as can realize the reduction of protein active.Disappearance region is longer can make gene inactivation conventionally more effectively.And, preferably, lack the reading frame difference of the upstream and downstream sequence in region.
The destruction of gene can also be moved frame sudden change by what for example introduce the sudden change (missense mutation), terminator codon (nonsense mutation) of aminoacid replacement, add to the coding region of gene on karyomit(e) or lack one or more Nucleotide, or similar approach (Journal of Biological Chemistry, 272:8611-8617 (1997); Proceedings of the National Academy of Sciences, USA, 955511-5515 (1998); Journal of Biological Chemistry, 26116,20833-20839 (1991)) realize.
The destruction of gene can also be realized by for example insert another sequence in gene coding region on karyomit(e).The site of inserting can be any region of gene, and the region of inserting is longer conventionally can make more effectively gene inactivation.Preferably, the reading frame of insertion point upstream and downstream sequence is different.Another sequence does not have particular restriction, as long as select the active sequence that can reduce or eliminate described coded protein, example comprises, for example, marker gene is as antibiotics resistance gene, to producing the useful gene of heterologous protein, etc.
As abovely can realize by for example following method the modification of gene on karyomit(e), prepare defective gene, wherein the partial sequence of gene is lacked, thereby make it can not produce the protein working orderly, with the recombinant DNA transform bacteria that contains this defective gene, make between gene, homologous recombination to occur on defective gene and karyomit(e), thereby with the gene on this defective gene substituted dyeing body.In this case, if comprise the marker gene of selecting according to host characteristics (as auxotroph) in recombinant DNA, operation can become easy.Even if the protein of being encoded by defective gene is produced, also there is the conformation different from wild-type protein, therefore its function is lowered or eliminates.This gene disruption based on utilizing the gene of homologous recombination to replace is established, and there is the method (Datsenko that is called " Red drives integration ", K.A, and Wanner, B.L., Proc.Natl.Acad.Sci.USA, 97:6640-6645 (2000)), use the method for linear DNA, for example utilize Red to drive the method (Cho of integration method and the combination from the system that cuts out of lambda particles phage, E.H., Gumport, R.I., Gardner, J.F., J.Bacteriol., 184:5200-5203 (2002)), the method of the plasmid that use contains responsive to temperature type replication orgin, use can conjugal transfer the method for plasmid, use the method (U.S. Patent No. 6 of the suicide vector without the starting point initial point of bringing into play function in host, 303, 383, the flat 5-007491 of Japanese Patent Application Laid-Open), Deng.
For the modification that reduces protein active can also realize by for example mutagenic treatment.The example of mutagenic treatment comprises common mutagenic treatment, for example x-ray irradiation or uv-radiation, and use mutagenic compound as the mutagenic treatment of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methane sulfonate (EMS) and methyl mesylate (MMS).
The reduction of target protein activity can be confirmed by the activity of measuring protein.The in the situation that of penicillin-binding protein, whether protein active has reduced can be passed through, and for example, measures transpeptidase activity and/or transglycosylase activity and confirms, determines according to the type under this protein.Transpeptidase and/or transglycosylase activity can be measured by for example method well known to the skilled person.Particularly, for example, the transpeptidase of PBP1a and transglycosylase activity can be turned to polysaccharide chains and be formed the crosslinked reaction of peptide by measurement lipid II oligomerization measures (Born P, et al., J Biol Chem.2006Sep15; 281 (37): 26985-93).Particularly, the activity decreased of protein to for example without modify observe in bacterial strain active 50% or lower, preferably 20% or lower, more preferably 10% or lower, more more preferably 5% or lower, or particularly preferably 0%.
The reduction that target gene is expressed can be by confirming that the minimizing of genetic transcription amount or the minimizing from the target protein quality of genetic expression confirm.
The minimizing of the target gene amount of transcribing can be by confirming the mRNA amount by genetic transcription and comparing of observing in not modified bacterial strain.The example that is used for the method for measuring mRNA amount comprises (Molecular Cloning, Cold spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001) such as Northern hybridization, RT-PCR.The amount of mRNA be preferably reduced to without modify observed amount in bacterial strain 50% or lower, 20% or lower, 10% or lower, 5% or lower, or 0%.
The reduction of target protein quality can be used and be confirmed (Molecular Cloning in conjunction with the antibody of this protein by Western trace, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA) 2001).The amount of protein is preferably reduced to, for example, without modify viewed amount in bacterial strain 50% or lower, 20% or lower, 10% or lower, 5% or lower, or 0%.
Depend on the means for destroying, the destruction of target gene can by determine gene partly or entirely, nucleotide sequence or the restriction endonuclease map of full-length gene etc. determine.
The above-mentioned method for reducing protein active can also be applied to any protein and their gene of coding after suitable variation, and for reducing the activity of penicillin-binding protein and the activity of reduction cell surface layer protein.
Hereinafter, by " for the gene construct of secreting, expressing heterologous protein " with make an explanation for the method that imports it.
Known, secretory protein is generally translated into front protein (also referred to as propetide) or front crude protein (also referred to as pre-pro-peptide), then by being processed into mature protein.Particularly, secretory protein is generally translated into front protein or front crude protein, then the signal peptide as fore portion (pre-part) by proteolytic enzyme (being generally called signal peptidase) excision, secretory protein changes mature protein or crude protein into thus.As for crude protein, its former part is further cut by proteolytic enzyme, thereby crude protein becomes mature protein.Therefore, in the method for the invention, preferably use signal peptide to be used for secreting generation heterologous protein.In the present invention, the front protein of secretory protein and front crude protein can be generically and collectively referred to as " secretory protein precursor ".In the present invention, " signal peptide " (also referred to as " signal sequence ") refers to and is present in secretory protein precursor N-end, and be not conventionally present in the aminoacid sequence in natural mature protein.
Although do not have particular restriction for gene construct of the present invention, as long as can realize the secretion of heterologous protein produces, but it preferably contains: the promoter sequence of bringing into play function in rod-like stem bacterial type bacterium, be connected to this promoter sequence downstream, be coded in rod-like stem bacterial type bacterium and bring into play the nucleotide sequence of the signal peptide of function, and be connected to the nucleotide sequence of this coded signal peptide downstream, coding heterologous protein nucleotide sequence.The nucleotide sequence of coded signal peptide can be connected to the downstream of promoter sequence, and signal peptide is expressed under the control of this promotor.The nucleotide sequence of coding heterologous protein can be connected the downstream of the nucleotide sequence of coded signal peptide, and this heterologous protein is expressed as and the fused protein of this signal peptide.Can also comprise for the regulating and controlling sequence (operator gene, terminator etc.) at rod-like stem bacterial type bacterium the Production of Heterologous Proteins plasmagene the correct position of described regulating and controlling sequence in performance function for gene construct of the present invention.
The present invention's promotor used does not have particular restriction, as long as select to bring into play the promotor of function in rod-like stem bacterial type bacterium, it can be promotor or the allogeneic promoter that is derived from rod-like stem bacterial type bacterium." in rod-like stem bacterial type bacterium, bring into play the promotor of function " and refer to the promotor that shows promoter activity in rod-like stem bacterial type bacterium.The specific examples of allogeneic promoter comprises, for example, and from colibacillary promotor for example tac promotor, lac promotor, trp promotor and araBAD promotor.Among them, strong promoter as tac promotor be preferred, inducible promoter as araBAD promotor be also preferred.
Example from the promotor of rod-like stem bacterial type bacterium comprises, for example, and the promotor of cell surface layer protein PS1, PS2 (also referred to as CspB) and SlpA (also referred to as CspA), and the promotor of various amino acid bio synthesis system genes.The specific examples of the promotor of various amino acid bio synthesis system genes comprises, the promotor of for example following gene: the gdh gene of L-glutamic acid biosynthesis system, the glutamine synthetase gene of glutamine synthesis system, the aspartokinase gene of Methionin biosynthesis system, the homoserine dehydrogenase gene of Threonine biosynthesis system, the acetohydroxy acid synthase gene of Isoleucine and α-amino-isovaleric acid biosynthesis system, the 2-isopropylmalate synthetase gene of leucine biosynthesis system, the Glutamate kinase gene of proline(Pro) and Arginine biosynthesis system, the phosphoribosyl-ATP pyrophosphorylase gene of histidine biosynthesis system, die aromatischen Aminosaeuren biosynthesis system is (as tryptophane, tyrosine, biosynthesis system with phenylalanine) deoxidation arabinose heptanone aldehydic acid phosphoric acid (DAHP) synthase gene, Nucleic acid system is as Phosphoribosyl tetra-sodium (PRPP) acyl transferase gene of t-inosinic acid and guanylic acid, imp dehydrogenase gene, with guanylic acid synthase gene.
About promotor, can utilize various report gene obtain the existing promotor of high reactivity type and use.For example, approach consensus sequence by-35 and-10th district that make promoter region, can improve the activity (International Patent Publication WO00/18935) of promotor.Paper (Prokaryotic promoters in biotechnology for assessment of the method for promotor intensity and strong promoter example at Goldstein etc., Biotechnol.Annu.Rev., 1,105-128 (1995)) etc. in have description.In addition, in known interval region between ribosome bind site (RBS) and translation initiation codon, particularly in the sequence (5'-UTR) of the direct upstream of initiator codon, replace, insert or lack several Nucleotide, can greatly affect the stability of mRNA and the translation efficiency of mRNA, therefore can modify this sequence.
The present invention's signal peptide used is not particularly limited, as long as select to bring into play the signal peptide of function in rod-like stem bacterial type bacterium, and it can be signal peptide or allos signal peptide from rod-like stem bacterial type bacterium." can in rod-like stem bacterial type bacterium, bring into play the signal peptide of function " and refer to following peptide, in the time that it is connected the N end of target protein, allow rod-like stem bacterial type bacterium to secrete this protein.Signal peptide is preferably as the signal peptide of the secreted protein of host's rod-like stem bacterial type bacterium, the more preferably signal peptide of rod-like stem bacterial type bacterial cell surface layer protein.The example of the cell surface layer protein of rod-like stem bacterial type bacterium comprises and is derived from the PS1 of corynebacterium glutamicum and PS2 (CspB) (the flat 6-502548 of Japanese patent application laid table) and the SlpA (CspA) (the flat 10-108675 of Japanese Patent Application Laid-Open) from corynebacterium ammoniagenes (C.stationis).The aminoacid sequence of PS1 signal peptide is as shown in SEQ ID NO:83, and the aminoacid sequence of PS2 (CspB) signal peptide is as shown in SEQ ID NO:84, and the aminoacid sequence of SlpA (CspA) signal peptide is as shown in SEQ ID NO:85.And, U.S. Patent No. 4,965,197 have described the signal peptide of existence from rod-like stem bacterial type DNA of bacteria enzyme, and these signal peptides also can be for the present invention.
Although signal peptide has some identical sequence signature between living species, in some living species, show that the signal peptide of secreting function might not show secreting function in another living species.Therefore,, in the time using allos signal peptide, can select suitably in rod-like stem bacterial type bacterium, to bring into play the signal peptide of function.Whether certain signal peptide brings into play function in rod-like stem bacterial type bacterium can be passed through, and for example, target protein and this signal peptide is expressed as to fused protein, and confirms whether this protein is secreted and add their confirmation.
Signal peptide can have the part n terminal amino acid sequence of the secretory protein in this signal peptide source.When the product of serving as interpreter is secreted cell, signal sequence is generally cut away by signal peptidase.In addition, as the encoding gene of signal peptide, although the gene of natural appearance can in statu quo use, also can modify it, thereby make it have the optimal codon of codon usage frequency that adapts to host used.
Secrete by the inventive method the example of heterologous protein producing and comprise, for example, biological activity protein, receptor protein, as antigen protein and the enzyme of vaccine.The example of enzyme comprises, for example, and trans-glutaminases, proteolytic enzyme, inscribe peptase, circumscribed peptase, aminopeptidase, carboxypeptidase, collagenase, chitinase etc.
The example of biological activity protein comprises, for example, and biotic factor, hormone, cytokine, antibody associated molecule.
The specific examples of somatomedin comprises, for example, Urogastron (EGF), rhIGF-1 (IGF-l), transforming growth factor (TGF), nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), vascular endothelial growth factor (VEGF), G-CSF (G-CSF), granulocyte-macrophage colony stimutaing factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), acid fibroblast growth factor (aFGF or fgf1), Prostatropin (bFGF or FGF2), epidermal keratinocyte somatomedin (KGF-1 or FGF7, with KGF-2 or FGF10), and pHGF (HGF).
The specific examples of hormone comprises, for example, and Regular Insulin, hyperglycemic-glycogenolytic factor, Somatostatin, human growth hormone (HGH), Rat parathyroid hormone 1-34 (PTH), and thyrocalcitonin.
The specific examples of cytokine comprises, for example, and interleukin-, Interferon, rabbit, tumour necrosis factor (TNFs).
The not strict differentiation each other of somatomedin, hormone and cytokine.For example, biological activity protein can be the protein that belongs to the single monoid of selecting from somatomedin, hormone and cytokine, or can be the protein that belongs to multiple monoids of selecting from above.
Biological activity protein can be whole protein, or can be a part for protein.The example of a part for protein comprises for example, having the part of physiologically active.The specific examples with the part of physiologically active comprises, for example, teriparatide (teriparatide), it is a kind of biologically active peptides, is made up of 34 amino-acid residues of Rat parathyroid hormone 1-34 (PTH) N end.
Antibody associated molecule refers to such protein, and it comprises the molecular species constituting by individual domain or two or more territories, and described territory is to select from the territory of complete antibody.The example in the territory of described complete antibody comprises VH, CH1, CH2 and CH3, and they are territories of heavy chain; With VL and CL, they are territories of light chain.Antibody associated molecule can be monomeric protein or polymer protein, as long as it comprises above-mentioned molecular species.In the situation that antibody associated molecule is polymer protein, antibody associated molecule can be the same polymer being made up of single a kind of subunit, or the heteromultimers being made up of two or more subunits.The specific examples of antibody associated molecule comprises, for example, and complete antibody, Fab, F (ab'), F (ab') 2, Fc, the dimer, Fc-fused protein, heavy chain (H chain), light chain (L chain), scFv (scFv), the sc (Fv) that are formed by heavy chain (H chain) and light chain (L chain) 2, disulfide linkage connect Fv (sdFv) and double antibody.
Receptor protein is not particularly limited, Ke Yishi, for example, the receptor protein of any biological activity protein and other biologically active substance.The example of other biologically active substance comprises, for example, and neurotransmitter, for example Dopamine HCL.In addition, receptor protein can also be the not yet certified orphan receptor of part.
The antigen protein that can be used as vaccine does not have special restriction, as long as it is the protein that can cause immune response, antigen protein can, according to the intention target mark of immune response, be selected suitably.
The specific examples of monomeric protein comprises, for example, and trans-glutaminases and type-1 insulin like growth factor (IGF-l).The example of transglutaminase gene comprises the secretion trans-glutaminases of following mushroom, if ray fungi is as Streptomyces mobaraensis (Streptoverticillium mobaraense) IFO13819, the verticillate bacterium of Chinese cassia tree chain (Streptoverticillium cinnamoneum) IFO12852, grey meat streptoverticillium (Streptoverticillium griseocarneum) IFO12776, streptomyces lydicus (Streptomyces lydicus) [WO9606931], filamentous fungus is as Oomycete [WO96/22366], etc.In addition, the specific examples of monomeric protein also comprises the monomeric protein as antibody associated molecule, for example heavy chain (H chain), light chain (L chain), scFv and sdFv.
Further, the specific examples of polymer protein comprises, for example, and vascular endothelial growth factor (VEGF), Regular Insulin, IL-5, interferon-γ, tumour necrosis factor (TNFs).In addition, the specific examples of polymer protein further comprises the polymer protein as antibody associated molecule, for example complete antibody, Fab, F (ab'), F (ab') 2, Fc, the dimer, Fc-fused protein, the sc (Fv) that are formed by heavy chain (H chain) and light chain (L chain) 2, and bivalent antibody.Among them, Fab, F (ab') 2, and Fc-fused protein be preferred.
Fab (fragment, antibodies) is the part except H LianFc district of complete antibody, and it is a kind of antibody fragment being only made up of antigen binding domain.Fab is the dimer being made up of the H chain Fab part of a molecule and the L chain of a molecule, the disulfide bonds that they are held by C.Complete antibody is the H2L2 tetramer, and has the heavy molecular weight of about 150kDa, and Fab has the small molecules amount of about 50kDa, and therefore Fab is considered to that destination organization is had to outstanding perviousness.Because Fab does not have Fc district, so it had not both had complement activity, there is no crystallizing power, but because it has antigen binding capacity yet, so it be mainly used in and the object of antigen.In antibody drug, Fab is noticeable especially in recent years.
F (ab') is the part outside complete antibody ChuFc' district and H chain.F (ab') is the dimer by molecule of H chain F (ab') part and a molecular composition of L chain, the disulfide bonds that they are held by C.In F (ab'), the remainder of H chain is longer than the remainder of H chain in Fab, therefore, in F (ab'), has retained the disulfide linkage part that connects H chain.Therefore, two molecules of F (ab') can form F (ab') by disulfide linkage 2.F (ab') and F (ab') 2also can be used as antibody drug, similar to Fab fragment.
Fc (fragment, crystallizable) is the antibody fragment that Jin You Fc district forms, and Fc district participates in complement activity and crystallizing power.Merged each other and the protein that forms is called Fc-fused protein by H Lian Fc district and another kind of functional protein.
The encoding gene of these protein can be modified according to host to be used, to obtain the activity of expectation.For example, the encoding gene of these protein be can modify, thereby interpolation that protein comprises one or more amino-acid residues, disappearance, replacement etc. made.The above-mentioned explanation about penicillin-binding protein and their gene of coding also can be applied to heterologous protein and the encoding gene thereof of secreting generation by the inventive method after suitable variation.Further, in the encoding gene of these protein, any codon all can replace with its equivalent cipher filial generation.For example, in the encoding gene of these protein, can, according to the codon frequency of observing in host, on demand codon be optimized.
N end regions by the final heterologous protein obtaining of the inventive method can be identical with natural protein, or can be different with natural protein.For example, the N end regions of the final heterologous protein obtaining can be the N end regions that comprises or the interpolation of several amino-acid residues or the natural protein of disappearance.Although the number of " one or several " amino-acid residue can change according to the total length of target heterologous protein or structure, particularly, it is preferably 1-20, more preferably 1-10, more more preferably 1-5.
Further, the heterologous protein that secrete generation can be the protein (crude protein) that contains primary structure part.Be crude protein in the case of secreting the heterologous protein of generation, the final heterologous protein obtaining can be crude protein or can not be crude protein.That is to say, crude protein can be formed to mature protein by excision primary structure part.Cutting can be by realizing with for example proteolytic enzyme.In the time using proteolytic enzyme, consider the activity of the protein that finally will obtain, general preferred at the position cutting crude protein substantially the same with natural protein, or more preferably, cut crude protein with the identical position of natural protein, thereby obtaining the mature protein identical with natural mature protein matter.Therefore, most preferably such specific protease, it,, at the cutting of position so crude protein, makes to produce the protein identical with naturally occurring mature protein.But the N-end regions of the heterologous protein that finally will obtain may be different from above-mentioned naturally occurring protein.For example, depend on type, the purposes etc. of the heterologous protein that will produce, compared with naturally occurring protein, increase or shorten the protein to several amino-acid residues at N end and may have more suitably active.Proteolytic enzyme available in the present invention comprises, for example, commercial available proteolytic enzyme, for example Dispase (being produced by Boehringer Mannheim), and can be from microbial culture medium, those that for example obtain in actinomycetic nutrient solution.These proteolytic enzyme can use under non-purified state, or also can after being purified to suitable purity as required, use.
Be not particularly limited for the method that gene construct used the present invention is incorporated into rod-like stem bacterial type bacterium.In bacterium of the present invention, may reside in the carrier (for example plasmid) of self-replicating outside karyomit(e) for gene construct of the present invention, or can group enter in karyomit(e).In addition, as mentioned above, in order to build bacterium of the present invention, can modify according to any order, for example, import for gene construct of the present invention, give or improve by secretion and produce the ability of protein, the activity of reduction penicillin-binding protein, the activity of reduction cell surface layer protein.
For for gene construct of the present invention, can use the carrier that for example contains this gene construct to be imported in host.Described carrier is not particularly limited, if select can be in rod-like stem bacterial type bacterium the carrier of self-replicating, carrier can be, for example, from the carrier of bacterial plasmid, from the carrier of yeast plasmid, from the carrier of phage, clay, phagemid etc.As carrier, for example, be preferred from the plasmid of rod-like stem bacterial type bacterium.Can in rod-like stem bacterial type bacterium, comprise pHM1519 (Agric.Biol.Chem., 48,2901-2903 (1984)) by the specific examples of the carrier of self-replicating; PAM330 (Agric.Biol.Chem., 48,2901-2903 (1984)); By improveing their plasmids that obtain, that there is drug resistance gene; The plasmid pCRY30 describing in the flat 3-210184 of Japanese Patent Application Laid-Open; Plasmid pCRY21, the pCRY2KE, pCRY2KX, pCRY31, pCRY3KE and the pCRY3KX that in the flat 2-72876 of Japanese Patent Application Laid-Open and U.S. Patent No. 5,185,262, describe; The plasmid pCRY2 and the pCRY3 that in the flat 1-191686 of Japanese Patent Application Laid-Open, describe; Plasmid pAJ655, the pAJ611 and the pAJ1844 that in the clear 58-192900 of Japanese Patent Application Laid-Open, describe; The pCG1 describing in the clear 57-134500 of Japanese Patent Application Laid-Open; The pCG2 describing in the clear 58-35197 of Japanese Patent Application Laid-Open; The pCG4 and the pCG11 that in the clear 57-183799 of Japanese Patent Application Laid-Open, describe; The pVK7 describing in the flat 10-215883 of Japanese Patent Application Laid-Open; The pVC7 describing in the flat 9-070291 of Japanese Patent Application Laid-Open; Etc..
Further, can also use artificial transposon etc.In the time using transposon, by the transposition ability of homologous recombination or transposon self, heterologous protein is incorporated in karyomit(e).Utilize other introducing method example of homologous recombination to comprise, for example: utilize linear DNA, have temperature sensitive replication origin plasmid, can conjugal transfer plasmid, do not there is the method that can bring into play the suicide vector of the replication origin of function in host, etc.In addition, in the time that heterologous protein plasmagene is introduced on karyomit(e), as long as be building up to karyomit(e) for gene construct of the present invention, any one in the nucleotide sequence of the promoter sequence comprising in gene construct and coded signal peptide or two can be originally to exist in host chromosome.Particularly, for example, by using as it is the promoter sequence of original existence in host chromosome and being connected in the nucleotide sequence of coded signal peptide in this promoter sequence downstream, and be only target heterologous protein plasmagene by the Gene Replacement in the nucleotide sequence downstream that is connected to coded signal peptide, also gene construct used the present invention can be building up on karyomit(e), build thus bacterium of the present invention.
In addition, in the situation that expressing two or more protein, bacterium of the present invention can be carried the each gene construct for the each protein of secreting, expressing, thus the secreting, expressing of realize target heterologous protein.Particularly, for example, the gene construct of the secreting, expressing protein that is useful on can be carried on single carrier, or can be carried on a karyomit(e).Further, can be carried at respectively multiple carriers for multiple gene constructs of secreting, expressing protein, or can be carried at respectively on single or multiple carriers and a karyomit(e)." express the situation of two or more protein " and comprise the situation that for example two or more heterologous proteins are produced in secretion, or the situation of heteromultimers protein is produced in secretion.
Be not particularly limited for the method that gene construct used the present invention is imported to rod-like stem bacterial type bacterium, and can use universal method, for example protoplasm body (Gene, 39,281-286 (1985)), electroporation (Bio/Technology, 7,1067-1070 (1989)), etc.
<2> is for generation of the method for heterologous protein of the present invention
The invention provides a kind of method for generation of heterologous protein, comprise and cultivate bacterium of the present invention and collect the heterologous protein (hereinafter also referred to as " method of the present invention " or " for generation of the method for heterologous protein of the present invention ") that secretion produces.
Bacterium of the present invention can be cultivated according to normally used method and condition.For example, bacterium of the present invention can be cultivated in the common substratum that contains carbon source, nitrogenous source and mineral ion.In order to obtain higher amplification, can add as required organic micro-nutrients, for example VITAMIN and amino acid.
As carbon source, can use such as dextrose plus saccharose of carbohydrate, organic acid for example acetic acid, alcohol and other.As nitrogenous source, can use ammonia, ammoniacal liquor, ammonium salt and other.As mineral ion, can use suitably as required calcium ion, magnesium ion, phosphate ion, potassium ion, iron ion etc.Cultivate in the OK range of pH5.0-8.5 and 15-37 DEG C, under aerobic conditions, carry out 1-7 days.Further, can use for producing the amino acid whose culture condition of L-by rod-like stem bacterial type bacterium, and other condition (with reference to WO01/23591 and WO2005/103278) of describing in use Sec type or Tat type signal peptide production method of protein.Further, in the time using inducible promoters for expressing heterologous protein, cultivation can also be undertaken by add promotor inductor in substratum.By cultivating under these conditions bacterium of the present invention, in cell, produce a large amount of target proteins, and be efficiently secreted into extracellular.In addition, the method according to this invention, the heterologous protein producing is secreted into cell outside, if be therefore accumulated in a large number may be lethal in microorganism cells protein, for example trans-glutaminases, also can produce and continuously without lethal effect.
The protein of secreting in substratum according to the inventive method can, after using method cultivation well known to the skilled person, separate and purifying from substratum.For example, after removing cell by centrifugal grade, can be by known appropriate method, for example saltout, ethanol precipitation, ultrafiltration, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium-pressure or high pressure liquid chromatography, reverse-phase chromatography and hydrophobic chromatography or these combinations, protein is separated and purifying.Further, in some cases, can use culture or culture supernatant according to former state.For secreting the protein in cell surface layer according to the inventive method, for example, by after well known to a person skilled in the art that method (improve salt concentrated and use tensio-active agent) is dissolved, also can by with protein secreting in addition separation and purifying of the identical mode of the situation in substratum.Further, in some cases, the protein of secretion in cell surface layer can be used as for example immobilized enzyme and uses, and without dissolving.
The secretion of target heterologous protein produces and can carry out SDS-PAGE as sample by the fraction that uses culture supernatant and/or contain cell surface layer, thereby confirms that the molecular weight of isolated protein band is confirmed.In addition, the secretion of target heterologous protein produce can be by using culture supernatant and/or contain cell surface layer fraction as sample, use antibody to carry out Western trace (Molecular Cloning, Cold spring Harbor Laboratory Press, Cold Spring Harbor (USA), 2001) confirmed.In addition, the secretion of target heterologous protein produces and can be confirmed by utilizing protein sequencer to measure N terminal amino acid sequence.Further, the secretion of target heterologous protein produces and can be confirmed by measure its quality with mass spectrograph.In addition, when target heterologous protein is enzyme or while having the bioactive protein that certain can measure, the secretion of target heterologous protein produce can be by using culture supernatant and/or contain cell surface layer fraction as sample, measure the biological activity of enzymic activity or protein and confirmed.
Embodiment
The present invention will further carry out concrete explanation with further reference to the following examples.But, should not think that these embodiment are in office where in the face of scope of the present invention causes restriction.
Embodiment 1: lack the structure (1) of corynebacterium glutamicum of penicillin-binding protein PBP1a and PBP1b for lacking the structure of carrier pBS Δ Cgl0278 of PBP1a encoding gene Cgl0278
The sequence of the Cgl0278 gene of the genome sequence of corynebacterium glutamicum ATCC13032 and Renicillin binding protein matter PBP1a is determined (Genbank accession number No.BA000036, NCBI gene entry NCgl0274).With reference to this sequence, synthesize the primer as shown in SEQ ID NO:01,02,03 and 04.Pass through PCR, use the chromosomal DNA (method [Biochim.Biophys.Acta of Saito and Miura of the corynebacterium glutamicum ATCC13869 bacterial strain of preparation in a conventional manner, 72,619 (1963)]) as template, with primer SEQ ID NO:01 and 02 and SEQ ID NO:03 and 04, the 5' side upstream region of the about 1kbp of Cgl0278 gene of the coding PBP1a that increased respectively and approximately the 3' side downstream area of 1kbp.Then, carry out PCR by the DNA fragmentation that uses these two amplifications as template, using the DNA as shown in SEQ ID NO:01 and 04 as primer, obtained by these two fragments and merged each other and the DNA fragmentation of about 2kbp of forming.In primer SEQ ID NO:01 and 04, be designed with respectively the recognition sequence of restriction enzyme BamH I and Xba I.PCR uses Pyrobest archaeal dna polymerase (Takara Bio production), and reaction conditions is the experimental program that manufacturers is recommended.Process this DNA fragmentation with restriction enzyme BamH I and Xba I, and insert in the BamH I-Xba I site of the pBS4 as described in WO2005/113744, obtained the carrier pBS Δ Cgl0278 for lacking Cgl0278 gene.Ligation is used DNA ligation kit Ver.2.1 (Takara Bio production), and reaction conditions is the experimental program that manufacturers is recommended.
(2) for lacking the structure of carrier pBS Δ Cgl2986 of PBP1b encoding gene Cgl2986
The sequence of the Cgl2986 gene of the genome sequence of corynebacterium glutamicum ATCC13032 and Renicillin binding protein matter PBP1b is by definite (Genbank accession number BA000036, NCBI gene entry NCgl0274).With reference to this sequence, synthesize the primer as shown in SEQ ID NO:05,06,07 and 08 according to the mode identical with Cgl0278.By using the chromosomal DNA of preparing from corynebacterium glutamicum ATCC13869 bacterial strain as template, with primer SEQ ID NO:05 and 06 and SEQ ID NO:07 and 08 carry out PCR, the 5' side upstream region of the about 1.3kbp of Cgl2986 gene of the coding PBP1b that increased respectively and approximately the 3' side downstream area of 1.1kbp.Then, by PCR, use the DNA fragmentation of these two amplifications as template, and DNA as shown in SEQ ID NO:05 and 08 is as primer, obtained by these two fragments and merged each other and the DNA fragmentation of about 2.4kbp of forming.The DNA fragmentation of about 2.4kbp of gained contains a recognition sequence for restriction enzyme Pst I and a recognition sequence for restriction enzyme Sal I.PCR uses Pyrobest archaeal dna polymerase (Takara Bio production), and reaction conditions is the experimental program that manufacturers is recommended.Process the fragment of an about 2.2kbp of DNA fragmentation acquisition above with restriction enzyme Pst I and Sal I, and be inserted in the Pst I-Sal I site of pBS5T, carrier, as described in WO2006/057450, has obtained the carrier pBS Δ Cgl2986 for lacking Cgl2986 gene.
(3) structure of PBP1a defect bacterial strain and PBP1b defect bacterial strain
Then, transform respectively with the pBS Δ Cgl0278 and the pBS Δ Cgl2986 that build the corynebacterium glutamicum YDK010 bacterial strain of describing in WO2004/029254.Corynebacterium glutamicum YDK010 bacterial strain is the cell surface layer protein PS2 defect bacterial strain (WO2004/029254) of corynebacterium glutamicum AJ12036 bacterial strain (FERM BP-734).According to method bacterium from the transformant of gained of WO2005/113744 and WO2006/057450 description, obtain the YDK010 Δ PBP1a bacterial strain of Cgl0278 genetic flaw and the YDK010 Δ PBP1b bacterial strain of Cgl2986 genetic flaw.Embodiment 2: the H chain region that uses respectively penicillin-binding protein PBP1a and PBP1b defective type corynebacterium glutamicum bacterial strain secreting, expressing antibody Herceptin (trastuzumab) Fab fragment
(1) for the structure of the plasmid of secreting, expressing antibody Herceptin Fab fragment H sequence
The gene order of mammary tumor cells specific antibody Herceptin H chain variable region is by definite (Genbank accession number No.AY513484).With reference to the sequence of this sequence and the non-variable region of universal antibody H chain, and consider the codon usage frequency of corynebacterium glutamicum to have synthesized the DNA as shown in SEQ ID NO:09-42.Use above-mentioned DNA as template, by PCR, increased in the total length H chain region of Herceptin as primer using the synthetic respectively DNA sequence dna as shown in SEQ ID NO:43 and 44, obtain the DNA fragmentation of the about 1.4kbp as shown in SEQ ID NO:45.The aminoacid sequence of the antibody Herceptin H chain of being encoded by SEQ ID NO:45DNA is as shown in SEQ ID NO:86.
Then, (pPKSPTG1 is for the carrier of secreting, expressing protransglutaminase (trans-glutaminases that contains primary structure part), contains: from the promotor of corynebacterium glutamicum ATCC13869 bacterial strain PS2 gene as template to use the pPKSPTG1 describing in WO01/23591; Can express be connected in this promotor downstream, coding is from the DNA of the signal peptide of corynebacterium ammoniagenes (C.stationis) ATCC6872 bacterial strain SlpA; And from the protransglutaminase gene of Streptomyces mobaraensis, this gene is connected and makes this protein as expressing with the fused protein of above-mentioned signal peptide), and primer as shown in SEQ ID NO:46 and 47, by pcr amplification the region that comprises aforementioned promoter region and aforementioned signal peptide district, obtain the DNA fragmentation of an about 0.7kbp.
Then, use the DNA fragmentation (fragment that comprises Herceptin total length H sequence and the fragment that comprises promotor and signal peptide district) of these two amplifications as template, using DNA as shown in SEQ ID NO:44 and 46 as primer, obtained the DNA fragmentation of an about 2.0kbp by PCR, it is merged each other and is formed by these two DNA fragmentations.
Then, use this fusion dna fragment as template, and as SEQ ID NO:46 and 48, SEQ ID NO:46 and 49, SEQ ID NO46 and 50, SEQ ID NO:46 and 51, SEQ ID NO:46 and 52, SEQ ID NO:46 and 53 and SEQ ID NO:46 and 54 as shown in DNA as primer, obtained the respectively DNA fragmentation for about 1.4kbp by PCR respectively.In primer SEQ ID NO:46, be designed with the recognition sequence of a restriction enzyme Kpn I.In each primer in SEQ ID NO:48,49,50,51,52,53 and 54, be designed with the recognition sequence of terminator codon and restriction enzyme Kpn I.PCR uses Pyrobest archaeal dna polymerase (Takara Bio production), and reaction conditions is the experimental program that manufacturers is recommended.Process these DNA fragmentations with restriction enzyme Kpn I, and they are inserted in the Kpn I site of the pPK4 as described in 9-322774 as flat in Japanese Patent Application Laid-Open separately, obtained can secreting, expressing Herceptin Fab part H sequence plasmid, pPKStrast-FabH (1-223C), pPKStrast-FabH (1-228T), pPKStrast-FabH (1-229C), pPKStrast-FabH (1-230P), pPKStrast-FabH (1-231P), pPKStrast-FabH (1-232C) and pPKStrast-FabH (1-233P).Particularly, utilize in these plasmids, can express the aminoacid sequence (numbering of effable amino-acid residue be included in plasmid title) of Herceptin H chain from 223,228,229,230,231,232 or 233 amino-acid residues of first amino-acid residue to the.By determining the nucleotide sequence that is inserted into fragment, confirm that the gene of expection has obtained structure.Definite use of nucleotide sequence terminator v3.1 cycle sequencing test kit (Applied Biosystems production), and 3130Genetic Analyzer (Applied Biosystems production).
(2) the H sequence of use penicillin-binding protein PBP1a deficient strain and PBP1b deficient strain secreting, expressing antibody Herceptin Fab fragment
By use the plasmid pPKStrast-FabH (1-229C) of the antibody Herceptin Fab fragment H sequence building in embodiment 2 (1), respectively YDK010 bacterial strain, YDK010 Δ PBP1a bacterial strain and YDK010 Δ PBP1b bacterial strain are transformed.By each transformant of gained in the MM liquid nutrient medium that contains 25mg/l kantlex (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0) cultivate 72 hours at 30 DEG C.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to reduced form SDS-PAGE, then use jewel orange (SYPRO Orange) (Invitrogen production) dyeing.Result, parent strain YDK010 and YDK010 Δ PBP1b bacterial strain do not detect target protein band, and only YDK010 Δ PBP1a bacterial strain detects a protein band (Fig. 1 and 2) identical with target antibody Herceptin Fab fragment H chain molecular weight.Use protein sequencer PPSQ-21A (Shimadzu production) to determine that the N terminal sequence of this band protein is visible, this sequence is consistent with the N terminal sequence of target antibody Herceptin Fab fragment H chain, the H chain of antibody Herceptin Fab fragment of therefore can having confirmed in culture supernatant secreting, expressing.
Then, by use each plasmid for secreting, expressing antibody Herceptin Fab fragment H sequence building in embodiment 2 (1), pPKStrast-FabH (1-223C), pPKStrast-FabH (1-228T), pPKStrast-FabH (1-229C), pPKStrast-FabH (1-230P), pPKStrast-FabH (1-231P), pPKStrast-FabH (1-232C) and pPKStrast-FabH (1-233P), transform YDK010 bacterial strain and YDK010 Δ PBP1a bacterial strain respectively.By each transformant of gained at the MM liquid nutrient medium that contains 25mg/l kantlex (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0) in 30 DEG C cultivate 72 hours.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to reduced form SDS-PAGE, then use jewel orange (Invitrogen production) dyeing.Result, in the time using any one secreting, expressing plasmid, the band of target protein in parent strain YDK010, all do not detected, and a protein band (Fig. 3) identical with target antibody Herceptin Fab fragment H chain molecular weight only in YDK010 Δ PBP1a bacterial strain, detected.
Embodiment 3: the L sequence that uses penicillin-binding protein PBP1a defective type corynebacterium glutamicum secreting, expressing antibody Herceptin Fab fragment
(1) for the structure of the plasmid of secreting, expressing antibody Herceptin Fab fragment L sequence
The gene order of mammary tumor cells specific antibody Herceptin L chain variable region is by definite (Genbank accession number No.AY513485).With reference to the sequence of this sequence and the non-variable region of universal antibody L chain, consider the codon usage frequency of corynebacterium glutamicum, synthesize the DNA as shown in SEQ ID NO:55-70.Use above-mentioned DNA as template, using the synthetic respectively DNA sequence dna as shown in SEQ ID NO:71 and 72 as primer, by PCR, increased in the total length L chain region of Herceptin, obtain the DNA fragmentation of an about 0.6kbp as shown in SEQ ID NO:73.The aminoacid sequence of the antibody Herceptin L chain of being encoded by SEQ ID NO:73DNA is as shown in SEQ ID NO:87.Then, use the pPKSPTG1 that describes in WO01/23591 as template (it contains from the promotor of corynebacterium glutamicum ATCC13869 bacterial strain with from the signal peptide district of corynebacterium ammoniagenes (C.stationis) ATCC6872 strain), and primer as shown in SEQ ID NO:74 and 75, by pcr amplification the region that comprises aforementioned promoter region and aforementioned signal peptide district, obtain the DNA fragmentation of an about 0.7kbp.Then, use the DNA fragmentation (fragment that comprises Herceptin L sequence and the fragment that comprises promotor and signal peptide district) of these two amplifications as template, DNA as shown in SEQ ID NO:74 and 76, as primer, has obtained the DNA fragmentation of an about 1.3kbp who is made up of these two DNA fragmentations that merge each other by PCR.In the primer of SEQ ID NO:74 and 76, be designed with the recognition sequence of restriction enzyme BamH I.PCR uses Pyrobest archaeal dna polymerase (Takara Bio production), and reaction conditions is the experimental program that manufacturers is recommended.Process this fusion dna fragment with restriction enzyme BamH I, and be inserted in the BamH I site of the pPK4 as described in 9-322774 as flat in Japanese Patent Application Laid-Open, obtained can secreting, expressing Herceptin Fab part L sequence plasmid pPKStrast-FabL.By determining the nucleotide sequence of Insert Fragment, confirm that the gene of expection has obtained structure.Use terminator v3.1 cycle sequencing test kit (Applied Biosystems production), and 3130Genetic Analyzer (Applied Biosystems production) has measured nucleotide sequence.
(2) the L sequence of use penicillin-binding protein PBP1a deficient strain secreting, expressing antibody Herceptin Fab fragment
By use the plasmid for secreting, expressing antibody Herceptin Fab fragment L sequence building in embodiment 3 (1), pPKStrast-FabL, transforms respectively YDK010 bacterial strain and YDK010 Δ PBP1a bacterial strain.By each transformant of gained at the MM liquid nutrient medium that contains 25mg/l kantlex (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0) in 30 DEG C cultivate 72 hours.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to reduced form SDS-PAGE, then use CBB R250 (Bio-Rad production) dyeing.As a result, YDK010 Δ PBP1a bacterial strain detects a protein band identical with target antibody Herceptin Fab fragment L chain molecular weight, and its band intensity is at least 2 times (Fig. 4) of the intensity observed of parent strain YDK010.In the time using protein sequencer PPSQ-21A (Shimadzu production) to determine the N terminal sequence of this band protein, sequence is consistent with the N terminal sequence of target antibody Herceptin Fab fragment L chain, the L chain of antibody Herceptin Fab fragment of therefore can having confirmed in culture supernatant secreting, expressing.
Embodiment 4: Fab (the H & L) fragment that uses penicillin-binding protein PBP1a defective type corynebacterium glutamicum secreting, expressing antibody Herceptin
(1) for the structure of the plasmid of secreting, expressing antibody Herceptin Fab (H & L) fragment
The DNA fragmentation (each is about 1.4kb) that the expression plasmid of the antibody Herceptin Fab fragment H sequence by building among embodiment 2 (1) with restriction enzyme Kpn I digestion is obtained is inserted in the Kpn I site of the expression plasmid pPKStrast-FabL of the antibody Herceptin Fab fragment L sequence of structure in embodiment 3 (1), obtained for the H sequence of coexpression Herceptin Fab fragment and the plasmid of L sequence: pPKStrast-FabH (1-223C)+L,
pPKStrast-FabH(1-228T)+L、pPKStrast-FabH(1-229C)+L、
pPKStrast-FabH(1-230P)+L、pPKStrast-FabH(1-231P)+L、
PPKStrast-FabH (1-232C)+L and pPKStrast-FabH (1-233P)+L.
(2) Fab (the H & L) fragment of use penicillin-binding protein PBP1a deficient strain secreting, expressing antibody Herceptin
By use among embodiment 4 (1) structure for secreting, expressing antibody Herceptin
The plasmid of Fab (H & L) fragment, pPKStrast-FabH (1-223C)+L,
pPKStrast-FabH(1-228T)+L、pPKStrast-FabH(1-229C)+L、
pPKStrast-FabH(1-230P)+L、pPKStrast-FabH(1-231P)+L、
PPKStrast-FabH (1-232C)+L and pPKStrast-FabH (1-233P)+L, transform respectively YDK010 bacterial strain and YDK010 Δ PBP1a bacterial strain.By each transformant of gained at the MM liquid nutrient medium that contains 25mg/l kantlex (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0) in 30 DEG C cultivate 96 hours.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to non-reduced type SDS-PAGE, then use jewel orange (Invitrogen production) dyeing, and compare the secretory volume of antibody Herceptin Fab (H & L) fragment.As a result, using when any one secreting, expressing plasmid, YDK010 Δ PBP1a bacterial strain compared with parent strain YDK010, the secretory volume of antibody Herceptin Fab (H & L) fragment be all significantly increased (Fig. 5).Use protein sequencer PPSQ-21A (Shimadzu production) to determine visible its N terminal sequence that comprises target antibody Herceptin Fab fragment H chain of N terminal sequence of Fab in the band detecting (H & L) protein and the N terminal sequence of L chain the transformant obtaining from YDK010 Δ PBP1a bacterial strain, therefore Fab (the H & L) fragment that can confirm antibody Herceptin has obtained expressing and secreting, thereby formed aggregate in culture supernatant.
(3) F (ab') of use penicillin-binding protein PBP1a deficient strain secreting, expressing antibody Herceptin 2fragment
Each the culture supernatant obtaining in embodiment 4 (2) is carried out to non-reduced type SDS-PAGE, then use gel Transfer Stacks PVDF, Mini (Invitrogen production) and iBlot tMgel transfer system (Invitrogen production) moves on to protein transduction on pvdf membrane.Using the Anti-Human IgG[H & L of alkali phosphatase enzyme mark] antibody (ROCKLAND production) and alkaline phosphatase put together substrate reagent box (Bio-Rad production) this pvdf membrane carried out to Western trace, detects the F (ab') of antibody Herceptin 2.As a result, detected and antibody Herceptin F (ab') carrying respectively for coexpression H sequence (it contains can form the Cys residue of disulfide linkage that connects each H chain) and the culture supernatant of plasmid pPKStrast-FabH (1-229C)+L, pPKStrast-FabH (1-230P)+L, pPKStrast-FabH (1-231P)+L, pPKStrast-FabH (the 1-232C)+L of L sequence and the transformant of pPKStrast-FabH (1-233P)+L 2the protein band that fragment molecular weight is identical.Further, even in the time using any one secreting, expressing plasmid, YDK010 Δ PBP1a bacterial strain is compared with parent strain YDK010, with antibody Herceptin F (ab') 2the band intensity of the protein that fragment molecular weight is identical be all significantly increased (Fig. 6).
Embodiment 5: the Fc fragment that uses penicillin-binding protein PBP1a defective type corynebacterium glutamicum secreting, expressing antibody Herceptin
(1) for the structure of the plasmid of secreting, expressing antibody Herceptin Fc fragment
DNA shown in the SEQ ID NO:45 of use synthetic total length H sequence that contains Herceptin in embodiment 2 (1) is as template, and use respectively synthetic as SEQ ID NO:77 and 78 and SEQ ID NO:77 and 79 as shown in DNA carry out PCR as primer, amplification Herceptin H Lian Fc district, has obtained the DNA fragmentation of each 0.7kbp of being approximately.Then, by using the pPKSPTG1 describing in WO01/23591 as template (it contains a promoter region from corynebacterium glutamicum ATCC13869 bacterial strain and a signal peptide district from corynebacterium ammoniagenes (C.stationis) ATCC6872 bacterial strain), use the primer as shown in SEQ ID NO:46 and 80, by pcr amplification the region that comprises aforementioned promoter region and aforementioned signal peptide district, obtained the DNA fragmentation of an about 0.7kbp.Then, use the DNA fragmentation (fragment that comprises Herceptin H sequence Fc district of these two amplifications, with each of the fragment that comprises promotor and signal peptide district) as template, as primer, obtain the DNA fragmentation of 1.4kbp that formed by above-mentioned two DNA fragmentations that merge each other respectively, about with DNA as shown in SEQ ID NO:46 and 77 by PCR.In the primer of SEQ ID NO:46 and 77, be designed with the recognition sequence of restriction enzyme Kpn I.PCR uses Pyrobest archaeal dna polymerase (Takara Bio production), and reaction conditions is the experimental program that manufacturers is recommended.Process these DNA fragmentations with restriction enzyme KpnI, then they are inserted into separately in the Kpn I site of the pPK4 as described in 9-322774 as flat in Japanese Patent Application Laid-Open, obtained can secreting, expressing Herceptin H sequence Fc district plasmid, pPKStrast-Fc (H224D-450) and pPKStrast-Fc (H231P-450).Particularly, utilize these plasmids, can express Herceptin H chain from the 224th or the 231st amino-acid residue to the 450th aminoacid sequence that amino-acid residue stops the numbering of express amino acid residue (can be included in plasmid title).The nucleotide sequence of the fragment of inserting by mensuration, confirms that the gene of expection has obtained structure.The mensuration of nucleotide sequence is used terminator v3.1 cycle sequencing test kit (Applied Biosystems production), and 3130Genetic Analyzer (Applied Biosystems production).
(2) the Fc fragment of use penicillin-binding protein PBP1a deficient strain secreting, expressing antibody Herceptin
By use the plasmid for secreting, expressing antibody Herceptin Fc fragment building in embodiment 5 (1), pPKStrast-Fc (H224D-450) and pPKStrast-Fc (H231P-450), transformed respectively YDK010 bacterial strain and YDK010 Δ PBP1a bacterial strain.By each transformant of gained in MM liquid nutrient medium (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0),, cultivate 72 hours at 30 DEG C.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to reduced form SDS-PAGE, then use gel Transfer Stacks PVDF, Mini (Invitrogen production) and iBlot tMgel transfer system (Invitrogen production) moves on to protein transduction on pvdf membrane.Using the Anti-Human IgG[H & L of alkali phosphatase enzyme mark] antibody (ROCKLAND productions) and alkaline phosphatase put together substrate reagent box (Bio-Rad production) this pvdf membrane carried out to Western trace, with the secretory volume of comparison antibody Herceptin Fc fragment.As a result, even in the time using any one secreting, expressing plasmid, YDK010 Δ PBP1a bacterial strain is compared with parent strain YDK010, and the secretory volume of antibody Herceptin Fc fragment also significantly improves (Fig. 7).
Embodiment 6: use penicillin-binding protein PBP1a defective type corynebacterium glutamicum secreting, expressing protransglutaminase
(1) use penicillin-binding protein PBP1a deficient strain secreting, expressing protransglutaminase
Utilize the protransglutaminase secreting, expressing system of corynebacterium glutamicum to have report (WO01/23591).Then,, by use the plasmid vector pPKSPTG1 for secreting, expressing protransglutaminase describing in WO01/23591, transform respectively YDK010 bacterial strain and YDK010 Δ PBP1a bacterial strain.By each transformant of gained at the MM liquid nutrient medium that contains 25mg/l kantlex (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0) in 30 DEG C cultivate 72 hours.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to reduced form SDS-PAGE, then use CBB R250 (Bio-Rad production) dyeing.Determine the secretory volume of protransglutaminase according to previous report (Protein Expr.Purif., 26:329-335), and this amount is compared.As a result, compared with the amount of observing in parent strain YDK010, in YDK010 Δ PBP1a bacterial strain, the secretory volume of protransglutaminase significantly improves (Fig. 8).
Embodiment 7: use the penicillin-binding protein PBP1a defective type corynebacterium glutamicum anti-digoxin single-chain antibody of secreting, expressing (scFv)
(1) for the structure of the plasmid of the anti-digoxin single-chain antibody of secreting, expressing (scFv)
The gene order of anti-digoxin scFv determined, and utilize subtilis to express to have exploration (Biotechnology (N Y)., 11 (1): 71-76 (1993)).With reference to this sequence, synthesize the DNA fragmentation as shown in SEQ ID NO:88 completely, this fragment comprises: from the promotor of corynebacterium glutamicum ATCC13869 bacterial strain PS2 gene; Can express be connected to this promotor downstream, coding is from the DNA of the expression signal peptide of the SlpA of corynebacterium ammoniagenes (C.stationis) ATCC6872 bacterial strain; With the DNA of the anti-digoxin scFv of coding, this DNA is connected and makes protein as being expressed with the fused protein of above-mentioned signal peptide.Synthetic DNA SEQ ID NO:88 holds at 5 ' end and 3 ' recognition site that comprises restriction enzyme Xba I.In synthetic DNA, the DNA of the anti-digoxin scFv that encodes considers that the codon usage frequency of corynebacterium glutamicum designs.In synthetic DNA, encode the nucleotide sequence of DNA of anti-digoxin scFv as shown in SEQ ID NO:89, and the aminoacid sequence of anti-digoxin scFv is as shown in SEQ ID NO:90.Digest this complete synthesis DNA fragmentation with restriction enzyme Xba I, and be inserted in the Xba I site of the pPK4 describing in JP9-322774A, acquisition can be expressed the plasmid of anti-digoxin scFv, pPKSSCA1.
(2) use the penicillin-binding protein PBP1a deficient strain anti-digoxin single-chain antibody of secreting, expressing (scFv)
By use the plasmid for the anti-digoxin scFv of secreting, expressing building in embodiment 7 (1), pPKSSCA1, transforms respectively YDK010 bacterial strain and YDK010 Δ PBP1a bacterial strain.By each transformant of gained at the MM liquid nutrient medium that contains 25mg/l kantlex (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0) in 30 DEG C cultivate 72 hours.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to reduced form SDS-PAGE, then use jewel orange (Invitrogen production) dyeing.As a result, a protein band identical with the anti-digoxin scFv molecular weight of target detected in YDK010 Δ PBP1a bacterial strain, its band intensity is than at least 2 times of the intensity height of observing in parent strain YDK010 (Fig. 9).In the time using protein sequencer PPSQ-21A (Shimadzu production) to determine the N terminal sequence of this band protein, sequence is consistent with the N terminal sequence of the anti-digoxin scFv of target, the anti-digoxin scFv that therefore can confirm in culture supernatant secreting, expressing.
Embodiment 8: Fab (the H & L) fragment that uses penicillin-binding protein PBP1a defective type corynebacterium glutamicum secreting, expressing antibody adalimumab (adalimumab)
(1) for the structure of the plasmid of secreting, expressing antibody adalimumab Fab (H & L) fragment
The aminoacid sequence of tumor necrosis factor-alpha (TNF-α) specific antibody adalimumab is by definite (assessment report (Assessment Report on February14 on February 14th, 2008,2008), medicine and medical instrument management office (Pharmaceuticals and Medical Devices Agency)).With reference to this sequence, synthesize the DNA fragmentation as shown in SEQ ID NO:91 completely, this fragment comprises: from the promotor of corynebacterium glutamicum ATCC13869 bacterial strain PS2 gene; Can express be connected to this promotor downstream, coding is from the DNA of the signal peptide of the SlpA of C.stationis ATCC6872 bacterial strain; And the 1st DNA to the aminoacid sequence of the 230th Cys residue of coding adalimumab H chain, this DNA is connected and makes this protein as being expressed with the fused protein of above-mentioned signal peptide; And further comprise in its downstream: from the promotor of corynebacterium glutamicum ATCC13869 bacterial strain PS2 gene; Can express be connected to this promotor downstream, coding is from the DNA of the signal peptide of the SlpA of corynebacterium ammoniagenes (C.stationis) ATCC6872 bacterial strain; With the DNA of L chain of coding adalimumab, this DNA is connected so that this protein is as being expressed with the fused protein of above-mentioned signal peptide.Similarly, synthesized the DNA fragmentation as shown in SEQ ID NO:92 completely, this fragment comprises: from the promotor of corynebacterium glutamicum ATCC13869 bacterial strain PS2 gene; Can express be connected to this promotor downstream, coding is from the DNA of the signal peptide of the SlpA of corynebacterium ammoniagenes (C.stationis) ATCC6872 bacterial strain; With the DNA of coding adalimumab L chain, this DNA is connected and makes this protein as expressing with the fused protein of above-mentioned signal peptide; And further comprise in its downstream: from the promotor of corynebacterium glutamicum ATCC13869 bacterial strain PS2 gene; Can express be connected to this promotor downstream, coding is from the DNA of the signal peptide of the SlpA of corynebacterium ammoniagenes (C.stationis) ATCC6872 bacterial strain; With the 1st DNA to the aminoacid sequence of the 230th Cys residue of coding adalimumab H chain, this DNA is connected and makes this protein as expressing with the fused protein of above-mentioned signal peptide.Synthetic DNA SEQ ID NO:91 and 92 all holds 5 ' recognition site that contains restriction enzyme BamH I separately, holds 3 ' recognition site that contains restriction enzyme Xba I.In these synthetic DNAs, the DNA of coding adalimumab H chain and L chain considers that the codon usage frequency of corynebacterium glutamicum designs.In these synthetic DNAs, the nucleotide sequence of the 1st DNA to the 230th amino acids sequence of coding adalimumab H chain is as shown in SEQ ID NO:93, and aminoacid sequence is as shown in SEQ ID NO:94.In addition, in these synthetic DNAs, the nucleotide sequence of the DNA of coding adalimumab L chain amino acid sequence is as shown in SEQ ID NO:95, and the aminoacid sequence of adalimumab L chain is as shown in SEQ ID NO:96.By restriction enzyme BamH I and Xba I digestion for the complete synthesis DNA fragmentation of each about 2.7kbp, and be inserted in the BamH I-Xba I site of the pPK4 describing in JP9-322774A, acquisition can coexpression adalimumab H chain (1-230C) and the plasmid of L chain, pPKSada-FabHL and pPKSada-FabLH." FabHL " in each plasmid title and " FabLH " have indicated the H chain gene of adalimumab and the group of L chain gene in expression plasmid to enter order.
(2) Fab (the H & L) fragment of use penicillin-binding protein PBP1a deficient strain secreting, expressing antibody adalimumab
By use the plasmid for secreting, expressing antibody adalimumab Fab (H & L) fragment building in embodiment 8 (1), pPKSada-FabHL and pPKSada-FabLH, transform respectively YDK010 bacterial strain and YDK010 Δ PBP1a bacterial strain.By each transformant of gained at the MM liquid nutrient medium that contains 25mg/l kantlex (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0) in 30 DEG C cultivate 96 hours.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to non-reduced type SDS-PAGE, then use jewel orange (Invitrogen production) dyeing, and compare the secretory volume of antibody adalimumab Fab (H & L) fragment.As a result, using when any one secreting, expressing plasmid, compared with parent strain YDK010, the secretory volume of antibody adalimumab Fab in YDK010 Δ PBP1a bacterial strain (H & L) fragment be significantly increased (Figure 10).Visible its of N terminal sequence that uses protein sequencer PPSQ-21A (Shimadzu production) to be determined at Fab (H & L) protein in the band detecting in the YDK010 Δ PBP1a bacterial strain transformant obtaining with pPKSada-FabHL conversion comprises the N terminal sequence of target antibody adalimumab Fab fragment H chain and the N terminal sequence of L chain simultaneously, therefore Fab (the H & L) fragment that can confirm antibody adalimumab has obtained expression and secretion, thereby formed aggregate in nutrient solution supernatant.Therefore visible, the same with the situation of expressing Herceptin Fab (H & L) fragment, in the situation that expressing adalimumab Fab (H & L) fragment, use penicillin-binding protein PBP1a defect bacterial strain can improve the secretory volume of monoclonal antibody (H & L) fragment.
Embodiment 9: the secreting, expressing of the structure of corynebacterium glutamicum ATCC13869 penicillin-binding protein PBP1a deficient strain and antibody Herceptin Fab (H & L) fragment
(1) structure of corynebacterium glutamicum ATCC13869 Δ PBP1a
Transform corynebacterium glutamicum ATCC13869 bacterial strain with pBS Δ Cgl0278, this carrier be among embodiment 1 (1) structure for lacking the carrier of gene of penicillin-binding protein PBP1a.From gained transformant, select bacterial strain according to the method described in WO2005/113744, obtain the ATCC13869 Δ PBP1a bacterial strain of Cgl0278 genetic flaw.
(2) by Fab (the H & L) fragment of corynebacterium glutamicum ATCC13869 Δ PBP1a secreting, expressing antibody Herceptin
By use among embodiment 4 (1) structure, for the plasmid of secreting, expressing antibody Herceptin Fab (H & L) fragment, pPKStrast-FabH (1-229C)+L, transforms respectively ATCC13869 bacterial strain and ATCC13869 Δ PBP1a bacterial strain.By each transformant of gained at the MM liquid nutrient medium that contains 25mg/l kantlex (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0) in 30 DEG C cultivate 96 hours.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to non-reduced type SDS-PAGE, then use jewel orange (Invitrogen production) dyeing, relatively the secretory volume of antibody Herceptin Fab (H & L) fragment.Result, compared with when using YDK010 bacterial strain as expressive host, in the time using ATCC13869 bacterial strain as expressive host, even if use penicillin-binding protein PBP1a defect bacterial strain, the secretory volume of antibody Herceptin Fab (H & L) fragment does not also improve (Figure 11 swimming lane 3 and 5).
Embodiment 10: the two structures of deficient strain of corynebacterium glutamicum ATCC13869 cell surface layer protein C spB and penicillin-binding protein PBP1a and the secreting, expressing of antibody Herceptin Fab (H & L) fragment
Corynebacterium glutamicum YDK010 bacterial strain increases because penicillin-binding protein PBP1a defect causes protein secreting amount, and it is cell surface layer protein PS2 (CspB) deficient strain (WO2004/029254) of corynebacterium glutamicum AJ12036 bacterial strain.Therefore, build the two defect bacterial strains of the CspB defect bacterial strain of ATCC13869 and the CspB of ATCC13869 and PBP1a, and carried out the secreting, expressing of antibody Herceptin Fab (H & L) fragment.The nucleotide sequence of the gene of coding ATCC13869 bacterial strain CspB is as shown in SEQ ID NO:97, and the aminoacid sequence of ATCC13869 bacterial strain CspB is as shown in SEQ ID NO:98.
(1) structure of corynebacterium glutamicum ATCC13869 Δ CspB and ATCC13869 Δ CspB Δ PBP1a
Use the chromosomal DNA (method [Biochim.Biophys.Acta, 72,619 (1963)] of Saito and Miura) of the corynebacterium glutamicum YDK010 bacterial strain of preparing in a conventional manner as template, SEQ ID NO:99 and 100 DNA carry out PCR as primer, the increased DNA fragmentation of an about 2.0kbp, it comprises the region that has CspB encoding gene defect.PCR uses Pyrobest archaeal dna polymerase (Takara Bio production), and reaction conditions is the experimental program that manufacturers is recommended.This DNA fragmentation is inserted in the Sma I site of the pBS5T describing in WO2006/057450, obtains the carrier pBS5T-Δ cspB for lacking cspB gene.
Then, transform corynebacterium glutamicum ATCC13869 bacterial strain with the pBS5T-Δ cspB building.From gained transformant, select bacterial strain according to the method for describing in WO2006/057450, obtain defective ATCC13869 Δ CspB bacterial strain in cspB gene.
Then, transform corynebacterium glutamicum ATCC13869 Δ CspB bacterial strain with pBS Δ Cgl0278, this carrier builds in embodiment 1 (1), for lacking the carrier of gene of penicillin-binding protein PBP1a.From gained transformant, select bacterial strain according to the method for describing in WO2005/113744, obtain equal defective ATCC13869 Δ CspB Δ PBP1a bacterial strain in cspB gene and Cgl0278 gene.
(2) by Fab (the H & L) fragment of ATCC13869 Δ CspB and ATCC13869 Δ CspB Δ PBP1a secreting, expressing antibody Herceptin
By use the plasmid for secreting, expressing antibody Herceptin Fab (H & L) fragment building in embodiment 4 (1), pPKStrast-FabH (1-229C)+L, transforms ATCC13869 Δ CspB bacterial strain and ATCC13869 Δ CspB Δ PBP1a bacterial strain respectively.By each transformant of gained at the MM liquid nutrient medium that contains 25mg/l kantlex (120g glucose, 3g magnesium sulfate heptahydrate, 30g ammonium sulfate, 1.5g potassium dihydrogen sulfate, 0.03g green vitriol, 0.03g seven water manganous sulfates, 450 μ g vitamins, 450 μ g vitamin Hs, 0.15g DL-methionine(Met) and 50g calcium carbonate, be dissolved in 1L volume water, be adjusted to pH7.0) in 30 DEG C cultivate 96 hours.After cultivation finishes, by each nutrient solution being carried out to centrifugal acquisition culture supernatant, and it is carried out to non-reduced type SDS-PAGE, then use jewel orange (Invitrogen production) dyeing, and compare the secretory volume of antibody Herceptin Fab (H & L) fragment.Result, compared with observing in parent strain ATCC13869, in the single defect strains A TCC13869 Δ CspB bacterial strain or ATCC13869 Δ PBP1a bacterial strain of CspB or PBP1a, the secretory volume of antibody Herceptin Fab (H & L) fragment does not improve, but, in two defect strains A TCC13869 Δ CspB Δ PBP1a, the secretory volume of antibody Herceptin Fab (H & L) fragment significantly improves (Figure 11).Therefore, show the two defect bacterial strains by using cell surface layer protein C spB and penicillin-binding protein PBP1a, can improve the secretory volume of monoclonal antibody (H & L) fragment.
Industrial applicibility
According to the present invention, provide a kind of and can efficiently secrete the rod-like stem bacterial type bacterium that produces heterologous protein.Further, the rod-like stem bacterial type bacterium that the application of the invention provides, as expressive host, can efficiently secrete generation heterologous protein, for example industrial useful protein.
Sequence table explanation
SEQ ID NO:01-08: primer
SEQ ID NO:09-42: for the DNA nucleotide sequence of completely synthetic Herceptin H chain
SEQ ID NO:43 and 44: primer
SEQ ID NO:45: the nucleotide sequence of Herceptin H chain encoding gene
SEQ ID NO:46-54: primer
SEQ ID NO:55-70: for the DNA nucleotide sequence of completely synthetic Herceptin L chain
SEQ ID NO:71 and 72: primer
SEQ ID NO:73: the nucleotide sequence of Herceptin L chain encoding gene
SEQ ID NO:74-80: primer
SEQ ID NO:81: the nucleotide sequence of corynebacterium glutamicum ATCC13032Cgl0278 gene
SEQ ID NO:82: by the aminoacid sequence of the protein of corynebacterium glutamicum ATCC13032Cgl0278 genes encoding
SEQ ID NO:83: the aminoacid sequence of corynebacterium glutamicum PS1 signal peptide
SEQ ID NO:84: the aminoacid sequence of corynebacterium glutamicum PS2 (CspB) signal peptide
The aminoacid sequence of SEQ ID NO:85:C.stationis SlpA (CspA) signal peptide
SEQ ID NO:86: the aminoacid sequence of Herceptin H chain
SEQ ID NO:87: the aminoacid sequence of Herceptin L chain
SEQ ID NO:88: for expressing the nucleotide sequence of complete synthesis DNA of anti-digoxin single-chain antibody
SEQ ID NO:89: the nucleotide sequence of anti-digoxin single-chain antibody encoding gene
SEQ ID NO:90: the aminoacid sequence of anti-digoxin single-chain antibody
SEQ ID NO:91 and 92: for expressing the nucleotide sequence of complete synthesis DNA of adalimumab Fab (H & L) fragment
SEQ ID NO:93: nucleotide sequence (coding region of 1-230C) the SEQ ID NO:94 of adalimumab H chain encoding gene: the aminoacid sequence (1-230C) of adalimumab H chain
SEQ ID NO:95: the nucleotide sequence of adalimumab L chain encoding gene
SEQ ID NO:96: the aminoacid sequence of adalimumab L chain
SEQ ID NO:97: the nucleotide sequence of corynebacterium glutamicum ATCC13869cspB gene
SEQ ID NO:98: by the aminoacid sequence of the protein of corynebacterium glutamicum ATCC13869cspB genes encoding
SEQ ID NO:99 and 100: primer

Claims (13)

1. there is the rod-like stem bacterial type bacterium that secretion produces the ability of heterologous protein,
This bacterium has reduced the activity of penicillin-binding protein through modifying, and in this bacterium, has reduced the activity of cell surface layer protein, and
Wherein said penicillin-binding protein is the protein with following character: in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of described heterologous protein increases than the secretion generation of observing in the bacterial strain of unmodified.
2. according to the rod-like stem bacterial type bacterium of claim 1, thus its through modification by reduction encode described penicillin-binding protein gene expression or destroy this gene and reduced the activity of described penicillin-binding protein.
3. according to the rod-like stem bacterial type bacterium of claim 1 or 2, wherein said penicillin-binding protein is PBP1a.
4. according to the rod-like stem bacterial type bacterium of any one in claim 1-3, wherein said penicillin-binding protein is (A) or (B) protein of middle definition below:
(A) there is the protein of the aminoacid sequence of SEQ ID NO:82,
(B) there is the aminoacid sequence of the SEQ ID NO:82 of the replacement, disappearance, insertion or the interpolation that comprise 1-10 amino-acid residue, and there is the protein of following character: in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of described heterologous protein increases than the secretion generation of observing in the bacterial strain of unmodified.
5. according to the rod-like stem bacterial type bacterium of any one in claim 1-4, thus its through modification by reduction encode described cell surface layer protein gene expression or destroy this gene and reduced the activity of described cell surface layer protein.
6. according to the rod-like stem bacterial type bacterium of any one in claim 1-5, wherein said cell surface layer protein is CspB.
7. according to the rod-like stem bacterial type bacterium of any one in claim 1-6, wherein said cell surface layer protein is (A) or (B) protein of middle definition below:
(A) there is the protein of SEQ ID NO:98 aminoacid sequence,
(B) there is the aminoacid sequence of the SEQ ID NO:98 of the replacement, disappearance, insertion or the interpolation that comprise 1-10 amino-acid residue, and there is the protein of following character: in the time that the activity of this protein in rod-like stem bacterial type bacterium is lowered, the secretion generation of described heterologous protein increases than the secretion generation of observing in the bacterial strain of unmodified.
8. according to the rod-like stem bacterial type bacterium of any one in claim 1-7, it is the bacterium that belongs to corynebacterium (Corynebacterium) or brevibacterium sp (Brevebacterium).
9. according to the rod-like stem bacterial type bacterium of any one in claim 1-8, it is corynebacterium glutamicum (Corynebacterium glutamicum).
10. according to the rod-like stem bacterial type bacterium of any one in claim 1-9,
Wherein said rod-like stem bacterial type bacterium has the gene construct for heterologous protein described in secreting, expressing, and
Wherein said gene construct comprises: the promoter sequence of bringing into play function in described rod-like stem bacterial type bacterium, be connected to this promoter sequence downstream, be coded in described rod-like stem bacterial type bacterium and bring into play the nucleotide sequence of the signal peptide of function, and be connected to nucleotide sequence nucleotide sequence downstream, that encode described heterologous protein of this coded signal peptide.
11. according to the rod-like stem bacterial type bacterium of any one in claim 1-10, and wherein said heterologous protein is antibody associated molecule.
12. according to the rod-like stem bacterial type bacterium of claim 11, and wherein this antibody associated molecule is by being selected from Fab, F (ab') 2, one or more protein in Fc-fused protein and scFv form.
13. 1 kinds of methods for generation of heterologous protein, it comprises cultivates according to the rod-like stem bacterial type bacterium of any one in claim 1-12, and collects the heterologous protein that secretion produces.
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